Targeted carriers for drug delivery across the gastrointestinal epithelium

ABSTRACT

A system and method for transcellular transport of compositions containing agents (e.g., research, analytical, reporter or molecular probes, diagnostic and therapeutic agents, biologically active agents, research agents, analytical agents, imaging agents, monitoring agents, enzymes, proteins, peptides, nucleic acids, lipids, sugars, hormones, lipoproteins, chemicals, viruses, bacteria, cells, including modified cells, biosensors, markers, antibodies and/or ligands) across the gastrointestinal epithelial layer including use of a composition containing the agent and a targeting moiety, specific for a determinant at the target location. An exemplary composition of the system includes an anti-ICAM antibody targeting moiety, specific for targeting ICAM-1. The system enables effective, versatile, and safe targeting and transport of agents. The system is useful in research applications, as well as in the context of translational science and clinical interventions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/463,796, filed May 3, 2012, which is a continuation in part ofU.S. application Ser. No. 13/376,362, filed Dec. 5, 2011, which is theNational Stage of International Application No. PCT/US2010/037490 filedJun. 4, 2010, which claims the benefit of U.S. Provisional ApplicationNo. 61/184,657 filed Jun. 5, 2009 and further claims the benefit of U.S.Provisional Application No. 61/220,404 filed Jun. 25, 2009. The benefitis further claimed of U.S. Provisional Application No. 61/481,779 filedMay 3, 2011. The disclosures of such applications are herebyincorporated by reference in their respective entireties, for allpurposes.

FIELD OF THE INVENTION

The present invention relates to novel ICAM-1-targeting moieties andmethods of using the same in compositions for targeted oral delivery andtransport of agents for research, analytical, diagnostic or therapeuticpurposes.

DESCRIPTION OF THE RELATED ART

Oral administration of nutritional supplements, therapeutics, and otheragents represents the most desirable route of delivery to the systemiccirculation (Owen, R. L. Semin. Immunol. 11 (1999): 157-163; Hidalgo, I.J. & Borchardt, R. T., Biochim. Biophys. Acta 1028 (1990): 25-30;Artusson, P., J. Pharma. Sci. 79 (1990): 476-482.). Whereas intravenousdelivery poses an inconvenience to healthcare professionals and causespatient discomfort due to its invasiveness, oral administration oftherapeutic agents includes various benefits, such as increased patientcompliance, reduction of cost, and a more flexible dosing regimen.However, in many cases, implementation of oral delivery needs toovercome crucial challenges of bioavailability (Chen, R. & Langer, R.,Adv. Drug Del. Rev. 34, 2-3 (1998): 339-350.). This concept refers tothe fraction of an administered drug that reaches the systemiccirculation, determined by numerous factors, including the degree ofgastrointestinal (GI) degradation, adhesion to the mucosa, and transportacross the GI tract.

An important obstacle of orally administered agents that influencestheir bioavailability is the extent to which they adhere to the mucosa.This event, referred to as bioadhesion, occurs as a prerequisite to GIabsorption. Substances must diffuse through the mucus layer, adhere tomucosal cells, and be transported across this cellular barrier into theblood (Ponchel, G. & Irache, J., Adv. Drug Del. Rev. 34 (1998): 191-219;Chen, R. & Langer, R., Adv. Drug Del. Rev. 34, 2-3 (1998): 339-350).Therefore, oral and GI delivery would benefit from strategies tosurmount these obstacles.

In an attempt to circumvent the challenges associated with oral drugdelivery and, most generally, with drug delivery via otheradministration routes, vehicles called carriers are currently beinginvestigated to assist and improve the bioavailability of therapeuticagents (Chen, R. & Langer, R., Adv. Drug Del. Rev. 34, 2-3 (1998):339-350; Bareford, L. & Swaan. P., Adv. Drug Del. Rev. 59(2007):748-758; Ghandehari, H. Adv. Drug Del. Rev. 60 (2008): 956; Kitchens, K.M., et al., Adv. Drug Del. Rev. 57 (2005): 2163-2176.). Carriers aremacromolecular assemblies fabricated from a variety of materials,designed to carry therapeutic or diagnostic agents. Functions ofcarriers include solubilization of hydrophobic drugs, protection ofdrugs against inactivation and premature activity en route to thetarget, optimization of a drug's pharmacokinetics (including circulationand tissue distribution), fine control of drug-release kinetics, andcontrol of drug metabolism and elimination. Key controllable parametersof carriers that define their utility for drug delivery include theirchemistry, surface characteristics, morphology, size, shape,permeability, biocompatibility, and biodegradability A great diversityof carriers has been designed with this purpose, including (but notrestricted to) nanotubes and other carbon nanostructures, linearpolymers, branched dendrimers, phospholipid liposomes, and amphiphilicpolymers formulated as self-assembled micelles or polymer particles(Torchilin, V., Adv. Drug Del. Rev. 58 (2006): 1532-1555; Ding, B. etal., Mol. Interventions. 6 (2006): 98-111; Lee, C. C., et al., Nat.Biotechnol. 23 (2005): 1517-1526; Ghandehari, H. Adv. Drug Del. Rev. 60(2008): 956; Minko, T., et al. Anticancer Agents Med. Chem. 6 (2006):537-552; Moghimi, S. & Szebani, J., Prog. Lipid Res. 42 (2003): 463-478;Hans, M. L. & Lowman, A. M., Curr. Opin. Solid State Mater. Sci. 6(2002): 319-327; Discher, B. M., et al., J. Phys. Chem. B. 106, 11(2002): 2848-2854; Panyam, J., & Labhasetwar, V., Adv. Drug Del. Rev. 55(2003): 329-347; Dziubla, T. D. & Muzykantov, V., V Torchilin, Ed.Imperial College Press, London. (2006): 499-506.)

Although such carriers optimize the bioavailability and pharmacokineticsof drugs in the blood and protect drugs from degradation in the GI andthe circulation, there still exist several problems associated with oraldelivery of carriers. For instance, most enterocytes (as most other celltypes) do not actively internalize and transport carriers across theirbody and, hence, across the GI epithelium, in a non-specific manner(Ponchel, G. & Irache, J., Adv. Drug Del. Rev. 34 (1998): 191-219.).Therefore, carriers which are not targeted to specific determinants inthe epithelial layer are sub-optimally absorbed by the GI tract giventheir low bioadhesion. Rather than adhering to the intestinal cells,non-specific carriers remain in the mucus layer where they becomedetached due to mucus turnover and elimination through the feces.Therefore, carriers hold potential for increasing the bioavailability oftherapeutic agents by protecting the drug from degradation in thegastrointestinal lumen (Ponchel, G. & Irache, J., Adv. Drug Del. Rev. 34(1998): 191-219; Ghandehari, H. Adv. Drug Del. Rev. 60 (2008): 956;Chen, H. et al., Pharm. Res. 13, 9 (1996): 1378-1383), yet efficientdelivery of carriers across the GI epithelium into the blood is stillsub-optimal.

The utility of carriers can be further optimized by coupling them tomolecules that have specific affinity for those sites in the body wherethe therapeutic agent needs to be delivered, which is referred to astargeted drug delivery. Coupling affinity-moieties (e.g., an antibody, anaturally-occurring ligand for the receptor or a functional derivativethereof, a vitamin, a hormone, a small molecule mimetic of anaturally-occurring ligand, a peptide, a polypeptide, a peptidomimetic,a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, acomponent of a microorganism, or any other molecule provided it bindsspecifically to the cell surface molecule) with affinity to certain cellsurface markers to the surface of carriers provides specific targeting,transport properties, and drug delivery capabilities. The concept ofselecting an affinity ligand to specifically recognize a molecularmarker or receptor can be applied to deliver therapeutics to a certainorgan, tissue, or subcellular compartment. Indeed, when injected in thecirculation, targeted carriers are capable of recognizingdisease-specific sites, which decreases side effects in healthy tissues(Ding, B. et al., Mol. Interventions. 6 (2006): 98-111; Chen, H. et al.,Pharm. Res. 13, 9 (1996): 1378-1383; Hamman, J. H., et al, Drug TargetInsights. 2 (2007): 71-81; Muro, S. & Muzykantov, V. R., Curr Pharm Des.11 (2005): 2383-2401.).

In the context of oral delivery, one advantage of targeted deliverysystems is that they delay intestinal excretion of drugs by enhancingbioadhesion. Intestinal epithelial cells present a vast array ofglycoproteins and glycolipids on the epithelial surface, which arereadily available to molecules present in the intestinal lumen. Thus,any molecules which possess binding affinity for these markers couldpotentially be used to direct carriers to the epithelium and therebyprolong the contact or transit time in the GI (Russell-Jones. G. J.,Adv. Drug Deliv. Rev. 46 (2001): 59-73; Chen, H. et al., Pharm. Res. 13,9 (1996): 1378-1383; Yin, Y. & Chen, D., J. Cont. Rel. 123, 1 (2007):27-38; Hamman, J. H., et al, Drug Target Insights. 2 (2007): 71-81.)

Significant progress in the identification of such cellular surfacemarkers-both relatively selective for GI epithelial cells and moregeneral molecules characteristic of several or many cell types in thebody—is being achieved using techniques including phage displaylibraries and monoclonal antibodies (Muzykantov, V. Expert Opin DrugDeliv 2 (2005): 909-926; Oh, P, et al, Nature 2004; 429:629-35.).However, the utility of most of these newly identified candidate markersfor drug delivery in humans remains to be tested. For example, functionsof most cell surface markers defined by these modern techniques areeither not known or are responsible for vital physiological processes inthe body, hence inadvertent intervention into or blocking of theirfunctions may lead to harmful side effects.

Targeted drug carriers are also more efficiently absorbed than theirnon-targeted counterparts because they may induce active transportpathways into and/or across cells. For example, carriers that targetvitamin B12 receptors enhance oral drug delivery compared to theirunconjugated counterparts, given that they utilize the B12 absorptionpathway in enterocytes, which operates via receptor-mediated endocytosis(Hamman, J. H., et al, Drug Target Insights. 2 (2007): 71-81.). Suchtransport of substances and pathogens into cells and/or across celllayers (e.g., endothelial cells in the blood-brain barrier andepithelial cells in the GI tract) involves either the transcellular orparacellular route. The paracellular pathway involves transport ofmolecules across the junctions that interlock epithelial cells. Thislateral domain of the epithelial barrier includes (i) the tightjunctions, a branching network of sealing strands mainly composed of theproteins occludins and claudins, and (ii) anchoring junctions known toas adherens junctions, which maintain cell-cell adherence by linkingtransmembrane proteins on adjacent cells to the cytoskeleton (Dejana, E.Nat. Rev. Mol. Cell Biol. 5 (2004): 261-270.). The paracellulartransport requires disturbance of these junctions, through whichmaterials are transported through the extracellular space. This may leadto the uncontrolled, passive and, hence, damaging transport ofsubstances other than the drug in the GI.

In contrast to the paracellular pathway, the transcellular route doesnot cause disruption of the permeability barrier and, hence, is bettersuited for safe and controlled drug delivery (Predescu, D., et al., Am.J. Physiol. Lung Cell Mol. Physiol. 287 (2004): L895-L901; Pardridge WM. Curr Opin Pharmacol 2006; 6:494-500.). This route involvesinternalization of materials on the apical membrane in contact with theGI lumen via membrane invagination, traffic of endocytic vesicles acrossthe enterocyte, and exocytosis at the basolateral membrane for deliveryinto the circulation (Predescu, D., et al., Am. J. Physiol. Lung CellMol. Physiol. 287 (2004): L895-L901; Pardridge W M. Curr Opin Pharmacol2006; 6:494-500.). This type of transport is mediated by endocytosis.The endocytic pathways include: (i) macropinocytosis, a mechanismallowing uptake of extracellular fluid into large micrometer sizevesicles, (ii) phagocytosis involves uptake of large particulate ligandsvia formation of large endocytic vesicles called phagosomes, (iii)clathrin-mediated endocytosis is triggered by binding of specificligands to their receptors in the plasma membrane, leading tointernalization of extracellular macromolecules along with extracellularfluid into vesicles coated by the cytosolic protein, clathrin(clathrin-coated pits), and (iv) caveolin-mediated endocytosis,characterized by uptake of materials into flask-shape vesicles enrichedon the protein caveolin-1, which occurs in areas of the membrane wherethe lipid bilayer is enriched in cholesterol and glycolipids (Muro, S.,et al. Curr. Vasc. Pharm. 2 (2004): 281-299.).

However, some of these natural pathways, e.g., macropinocytosis andphagocytosis, are typically associated to cells of the immune system,precluding targeting and delivery to other cell types in the body (Muro,S., et al. Curr. Vasc. Pharm. 2 (2004): 281-299.). In addition, allthese pathways have been found to be suppressed in certain types ofhuman pathology including inflammation, metabolic disorders, ischemiaand abnormalities of blood flow, negatively impacting delivery in thesetting in which drugs are needed in the tissues (Dermaut B, et al. JCell Biol 2005; 170:127-39; Dhami R, & Schuchman E H. J Biol Chem 2004;279:1526-32; Monroy M A, et al. Bone 2002; 30:352-9; Pol A, et al. MolBiol Cell 2005; 16:2091-105; Puri V, et al. Nat Cell Biol 1999;1:386-8.).

Also, intracellular and transcellular transporting capacity of clathrin-and caveolar-mediated pathways common to most cell types in the body(e.g., exploited by targeting to manose-6-phosphate receptor, glucosereceptors, LDL-family receptors, receptor associated protein RAP,insulin-like growth factor II, transferrin, insulin, folate receptor,and other receptors), is restricted to relatively small objects,typically <100 nm in diameter (Muro, S., et al. Curr. Vasc. Pharm. 2(2004): 281-299; Pardridge W M. Curr Opin Pharmacol 2006; 6:494-500;Schnitzer J E, Adv Drug Deily Rev 2001; 49:265-80.). For instance, phageparticles (˜800 nm length) targeted to pulmonary caveolar determinantssimply do not bind to their intended targets due to inaccessibility ofcaveolar determinants for objects larger than 50-80 nm, from thecirculation (Oh et al., 2007) This fact restricts transport of manyemerging targeted drug and diagnostic delivery systems (100 nm-1 μm)with promising applications in virtue of their high affinity andpayload.

Finally, even if these obstacles are overcome, the transported carriersmust be able to then transport their cargoes (therapeutics and/ordiagnostic agents) through the circulation to the different organs,tissues, cell types, and subcellular compartments in the body wheretheir action is required. Therefore, such carriers must be targeted tomarkers that are present not only on GI epithelial cells but also cellsin the blood vessel wall and cells within the different tissues andorgans in the body, primarily on sites affected by disease. Ultimately,safe targeting moieties much be available to target such carriers acrossthe GI epithelium and to all these body destinations in a safe manner,e.g., relatively “invisible” and innocuous to the body to avoidsecondary detrimental reactions, particularly if recurrentadministrations are necessary for an effective treatment.

There therefore remains a need in the art for targeting moieties andcompositions comprising such targeting moieties for safe and controlleddelivery via the gastrointestinal tract, where the targeting moietieseffectively target GI epithelial cells, and are transported across theGI epithelial layer with no effect on the GI permeability and furtherprovide systemic availability of the composition. The present inventionprovides such methods and compositions.

SUMMARY OF THE INVENTION

This invention relates to the use of targeting moieties effective astargeting molecules providing efficient and specific binding ofcompositions containing therapeutic agents and drug delivery systems toa determinant present in both mice and humans, e.g. for delivery to thesurface of a cell and/or effective and safe transport into and/or acrosscells. In one aspect the targeting moieties are short peptides derivedfrom fibrinogen, a natural protein present in the human circulation. Inanother aspect the targeting moieties are anti-ICAM-1 antibodies.

The present invention relates to a composition for oral administrationto a subject, the composition comprising a targeting moiety comprisingan anti-ICAM antibody; and an agent, wherein the targeting moietyrecognizes and binds to ICAM-1 on a GI epithelial cell and thecomposition is transported across the GI epithelium. In various aspects,the composition may further comprise a delivery carrier, a protectiveagent and/or a second or additional targeting moiety.

In another aspect the invention relates to a method for delivery of anagent across the gastrointestinal epithelium, comprising oraladministration of a composition comprising a targeting moiety selectedfrom SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and an anti-ICAMantibody; and an agent, wherein the targeting moiety recognizes andbinds to a target on a gastrointestinal epithelial cell and thecomposition is transported across the gastrointestinal epithelium.

A further aspect of the invention relates to a targeting moiety specificfor targeting ICAM-1, the targeting moiety selected from SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and an anti-ICAM antibody.

Other aspects, features and embodiments of the invention will be morefully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of Example 1, comparing the in vivoaccessibility and internalization in cells, of free anti-ICAM andanti-ICAM carriers, in vivo and in cell cultures, in normal and diseaseconditions, via ICAM-1 and CAM-mediated endocytosis, as compared toendocytosis mediated by clathrin, caveoli, macropinocytosis andphagocytosis.

FIG. 2 illustrates the results of Example 2, where FIG. 2A-2C providefluorescence and electron microscopy images of ICAM targeting by ICAM-1specific targeting moieties and FIG. 2D is a graph of location of thecarriers, as a function of their size.

FIG. 3 illustrates the results of Example 3, showing efficient targetingand internalization of various delivery compositions and various celltypes.

FIG. 4 illustrates the results of Example 4, where FIG. 4A provides agraph of the relative binding of γ3 carriers to both human and mouseICAM-1, and as compared to anti-ICAM carriers; FIG. 4B provides a graphdemonstrating the effects of blocking ICAM-1 or other molecules on thebinding of γ3 carriers with ASM cargo and binding to cells that do notexpress ICAM-1; FIGS. 4C-E provide graphs of the lysosomal transport asa function of time, peptide type (γ3, 2γ3 and 3γ3) and variousinhibitions and activations.

FIG. 5 illustrates the results of Example 5, demonstrating efficient andspecific binding of various ICAM-1-targeting moieties or virusesexpressing these moieties to human and mouse ICAM-1 via peptides A1, B7,B8, B9, B10 and D6 as targeting moieties.

FIG. 6 illustrates the in vivo pharmokinetics and biodistribution of γ3,2γ3 and 3γ3 targeting moieties on carriers targeting therapeutic agentstargeting ICAM-1, as provided in Example 6.

FIG. 7 illustrates the ICAM-1-targeting ASM plasmid (B), as utilized inExample 8, where the plasmid contains a coding sequence for any of γ3,derivatives of γ3 (e.g., 2γ3 or 3γ3), A1, B7, B8, B9, B10, and D6.

FIG. 8 is transmission electron microscopy images of the in vivo braintransport of compositions of the invention, as described in Example 9hereof: (A) demonstrates delivery compositions bound to the surface ofor endocytosed within vascular endothelial cells in the blood-brainbarrier; and (B) demonstrates delivery compositions that have migratedacross the blood-brain barrier into the brain.

FIG. 9 is a graphical illustration of inhibition of leukocytetransmigration across endothelial cells by various ICAM-1-targetingpeptides, as described in Example 10 hereof.

FIG. 10(A) is a graphical illustration of the amount of microemboli incirculation in blood, as evaluated at 1 min after injection and FIG.10(B) is a graphical illustration of the amount of microemboli lodgingin the vasculature of the kidneys and spleen at 15 min after injection,both as described in Example 11 herein.

FIG. 11 is microscopy images of ICAM-1 expression in Caco-2 cells, asdescribed in Example 12 below.

FIG. 12(A) provides fluorescence microscopy images of IgG (control) andanti-ICAM carriers to Caco-2 cells, FIG. 12(B) is a graphicalillustration of the number of carriers bound per cell and FIG. 12(C) isa graphical illustration of the results of inclusion of free anti-ICAMantibodies in solution, as described in Example 13.

FIG. 13 provides graphs of the kinetics of binding and endocytosis ofICAM-1 targeted carriers in Caco-2 cells, as described in Example 14.

FIG. 14 provides (A) fluorescence microscopy images and (B) graphicanalysis of the internalization of ICAM-1 targeted carriers in Caco-2cells, as described in Example 15.

FIG. 15 provides fluorescence microscopy images visualizing thejunctions between Caco-2 cells, as correlated with TEER values inExample 16.

FIG. 16 provides fluorescence microscopy images confirming the (A)expression of ICAM-1 in Caco-2 cells, and (B) absence of expression ofPECAM-1 as described in Example 17.

FIG. 17 provides graphs of the transport of ICAM-1 targeting carriersacross Caco-2 monolayers, as described in Example 18.

FIG. 18 provides graphs of the rate of carrier transport across Caco-2cells, as described in Example 19.

FIG. 19 provides graphs of the transport of anti-ICAM-1 carriers acrossCaco-2 cells, as described in Example 20.

FIG. 20, provides graphs of the CAM-mediated transport ofICAM-1-targeting moieties, as described in Example 21.

FIG. 21 provides illustrations of (A) protection of anti-ICAM carriersfrom degradation, (B) release of protected carriers, and (C) comparisonof endocytic versus paracellular transport across the GI epitheliallayer.

FIG. 22 provides an illustration of a polymer coated carrier, protectedfrom detection by the immune system.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The present invention relates to targeting moieties used to provide moreeffective, versatile, and safe targeting and transport of molecularprobes, diagnostic and therapeutic agents and/or their carriers. Themoieties provided are useful in both research settings and in thecontext of translational science and clinical interventions. Theinvention further relates to compositions containing such targetingmoieties and to methods of using such target moieties.

“Targeting moiety” as used herein refers to moieties useful in thecompositions and methods described herein, wherein the moietiesspecifically target a surface expressed protein, e.g., ICAM-1. In oneembodiment the targeting moieties mediate efficient and specificdelivery of an agent to a determinant through such targeting. Targetingmoieties and/or their carriers may be varied, such as by size, shape,and/or valency in order to modulate delivery. A targeting moiety mayalso be described herein as any of an “affinity moiety,” an “affinitypeptide,” a “targeting peptide,” “targeting molecule,” or “ligand.”

“Carrier” as used herein refers to an accessory substance present as avehicle during the transfer of an active substance to a target. In oneembodiment, the targeting moieties are carriers for the agents. As such,the targeting moieties may also be referred to herein as “targetingcarriers.” In another embodiment, “delivery carriers” are provided fortransfer or delivery of a composition containing the targeting moietyand/or the agent to the target. A delivery carrier may also function toprotect the targeting moiety and/or the agent between the time ofadministration and the time of arrival at the locus of the target. Insuch an embodiment the delivery carrier may alternately be referred toas a “protective agent.”

“Agent” as used herein refers to the subject of the targeting ortransport of the targeting moiety. In conjunction with the action of thetargeting moiety, agents may be transported to a cell, to the surface ofa cell, into a cell or across a cell. Specifically, the targeting moietyprovides a means for transporting agents such as, but not limited to,research, analytical, reporter or molecular probes, diagnostic andtherapeutic agents, biologically active agents, research agents,analytical agents, imaging agents, monitoring agents, enzymes, proteins,peptides, nucleic acids, lipids, sugars, hormones, lipoproteins,chemicals, viruses, bacteria, cells, including modified cells,biosensors, markers, antibodies and ligands. An agent may also bedescribed herein as a “cargo.”

“Target” as used herein refers to a determinant recognized by thetargeting moiety. In one embodiment a target recognized by thistargeting moiety is a molecule expressed on the surface of a cell whichconstitutes a part of the first layer after administration within atissue. Such cells are therefore easily accessible. Examples of suchcells include, but are not limited to, endothelial cells in the contextof preferable intravascular (intravenous or intra-arterial)administration; epithelial cells in the case of gastrointestinal, nasal,intratracheal, or rectal administration; immune system cells fortreatment of conditions affecting these cells; muscle cells forintramuscular administration; glial cells and neurons for intra-cerebralor intrathecal administration and the like. Expression of the targetmolecule in normal physiological conditions would provide a target forprotective or prophylactic interventions, whereas up-regulation of theexpression of the molecule in many pathologies would provide a targetfor site-specific interaction with the composition, such as delivery oftherapeutic and diagnostic agents to disease sites and/or cell-specifictransport of the composition, including, but not limited to surfaceresidency, intracellular transport and transcellular transport of thecomposition and/or agent. In another embodiment a target recognized bythe targeting moiety may be a tissue or an organ. A target may also bedescribed herein as a “target molecule,” a “determinant,” or a“receptor.”

Where the target molecule is expressed on the surface of a cell, itshould be stably expressed on the cell surface to allow sufficient timeframe for targeted interventions. If the cell surface is the intendeddestination for a composition or agent, the target molecule shouldremain on the cell surface. If the interior of the cell is the intendeddestination for a composition or agent, the target molecule shouldprovide internalization within the cell body or safe transport acrosscellular layers (e.g., by an endocytic pathway or transcytosis,respectively) upon proper induction by targeting. Such internalizationmay be used for intracellular delivery of agents to the cell interior ortranscellular delivery for penetration across cell layers or tissues. Ina particular embodiment the invention provides a method of CAM-mediatedcytosis of a composition across the GI epithelial layer.

The physiological function of the target molecule is not detrimental(and, preferably is beneficial) to the interaction of the agent with thetarget molecule and/or the biological, physiological, or pathologicalfunction of the target molecule. The mechanism associated withinteraction of the agent and the target molecules resulting in any ofsurface residency, intracellular transport or transcellular transportshould be not affected by disease, and the parameters of suchinteraction should be known, to allow rational design of strategies fordelivery of therapeutic and/or diagnostic agents with precision.

In one embodiment the invention provides a composition including atargeting moiety, where the targeting moiety recognizes and binds to atarget on a cell. A composition of the invention may optionally includetwo or more targeting moieties, where the two or more targeting moietiesmay react with their targets simultaneously or sequentially but,preferably, do not compete for a particular target. Optionally, thecomposition may further comprise a delivery carrier. Still further, thecomposition may optionally comprise a protective agent to protect thecomposition e.g., from degradation in the digestive system or fromdetection by the immune system. In another embodiment the compositionincludes a nucleic acid encoding a targeting moiety, where the targetingmoiety, when expressed, recognizes and binds to a target on a cell.

In still another embodiment the invention provides a composition fordelivery of an agent to a cell, where the delivery composition includesboth a targeting moiety and one or more agents, where the targetingmoiety recognizes and binds to a target on a cell and is effective todeliver the agent to the cell. Optionally, the composition may furthercomprise a delivery carrier. In a further embodiment, the inventionprovides a composition for delivery of an agent to a cell, where thedelivery composition includes both a nucleic acid encoding a targetingmoiety and a nucleic acid encoding an agent, where the targeting moiety,when expressed, recognizes and binds to a target on a cell and iseffective to deliver the agent to the cell. Expression of the agent maybe co-expression with the targeting moiety or may be separatelyexpressed.

Expression and production systems as described above may include, butare not limited to, viruses, bacteria, and eukaryotic cells expressingand producing the targeting moieties and/or the agents.

In an exemplary embodiment, the inventors explored the utility as atargeting moiety of a 17 amino acid peptide derived from the γ chain offibrinogen and its cleavage product fibrin, known as γ3, (Altieri D. C.,et al., J. Biol. Chem. 1995; 270:696-9; D'Souza S. E., et al., J. Biol.Chem. 1996; 271:24270-7; Duperray A., et al., J. Biol. Chem. 1997;272:435-41; Languino, L. R., et al., Cell 1993; 73:1423-34.) and twopeptides derived from γ3, derivatives termed 2γ3 and 3γ3. Fibrinogen isa dimer of three pairs of disulfide-bonded chains (Aα, Bβ, and γ)arranged in three globular domains (a central E domain and twoperipheral D domains). (Weisel J. W., Adv. Protein Chem. 2005;70:247-99.) Cleavage by thrombin of fibrinopeptides A and B from theamino-terminus of chains Aα and Bβ, converts fibrinogen into fibrin andexposes polymerization sites for forming fibrin mesh, typically involvedin blood clotting. Both fibrinogen and fibrin form a molecular bridgebetween endothelial ICAM-1 and leukocyte β2 integrin Mac-1, which is themain function attributed to ICAM-1 in regard to fibrinogen/fibrininteraction: strengthening of the adhesion between these two cell typesduring inflammation.

Although the γ3 sequence within the fibrinogen/fibrin γ chain has beendemonstrated to bind to human ICAM-1, none of native fibrinogen, fibrin,the γ chain, or the γ3 peptide had been coupled to or tested in thecontext of targeting therapeutic or diagnostic agents or their carriersto ICAM-1, or any other cell surface determinant, prior to the presentinvention. In addition, they had not been observed to affect cellulartransport, for example internalization by cells by induction ofCAM-mediated endocytosis or by any other method, and the potentialrecognition of ICAM-1 in other species (e.g., mouse) by the γ3 peptidewas unknown. Prior to the inventors' work, potential use of γ3 for safeand efficient ICAM-1 targeting and delivery of therapeutic anddiagnostic compounds, and their carriers, was unpredictable andunlikely.

γ3, 2γ3 and 3γ3 were examined as targeting moieties with regard totargeting therapeutic agents to the surface molecule “intercellularadhesion molecule 1” (ICAM-1). Therefore, in one embodiment of theinvention, the targeting moiety is selected from γ3 (amino-terminusNNQKIVNLKEKVAQLEA carboxyl-terminus (SEQ ID NO: 1)), 2γ3 (amino-terminusNNQKIVNIKEKVAQIEA carboxyl-terminus (SEQ ID NO: 2)), and 3γ3(amino-terminus NNQKLVNIKEKVAQIEA carboxyl-terminus (SEQ ID NO: 3)),short peptides derived from fibrinogen, a natural protein present inhuman circulation, which retain their affinity and selectivity forparticular target determinants, particularly ICAM-1.

As can be seen from SEQ ID NOs: 2 and 3, peptides 2γ3 and 3γ3 arederivatives of γ3. Peptides 2γ3 and 3γ3 both retain the affinity andselectivity for ICAM-1, as demonstrated by γ3. In another embodiment ofthe invention, the targeting moiety is an additional variant orderivative of γ3, wherein the variant or derivative is truncated orextended with respect to SEQ ID NO: 1 and/or contains one or more aminoacid substitutions, deletions, insertions, and/or additions relative toSEQ ID NO: 1. Such a targeting moiety retains affinity and selectivityfor particular target determinants, such as ICAM-1. Accordingly,targeting moieties as described herein may include peptidomimetics ofany of γ3, 2γ3, or 3γ3 their nucleotide encoding sequences, and theviral, bacterial, or cellular systems expressing and/or producing thesepeptides.

In a further embodiment, the inventors explored the utility as atargeting moiety of various very short chain peptides developedutilizing phage display. From such investigation a battery of 7 aminoacid peptides (termed A1, B7, B8, B9, B10 and D6) was determined to beindividually useful as targeting moieties in targeting of ICAM-1.Therefore, in one embodiment of the invention, the targeting moiety isselected from A1 (amino-terminus YPASYQR carboxyl-terminus (SEQ ID NO:14)), B7 (amino-terminus YQATPLP carboxyl-terminus (SEQ ID NO: 15)), B8(amino-terminus GSLLSAA carboxyl-terminus (SEQ ID NO: 16)), B9(amino-terminus FSPHSRT carboxyl-terminus (SEQ ID NO: 17)), B10(amino-terminus YPFLPTA carboxyl-terminus (SEQ ID NO: 18)), D6(amino-terminus GCKLCAQ carboxyl-terminus (SEQ ID NO: 19)), phagedisplay-derived peptides. In another embodiment, the targeting moiety isan additional variant or derivative of any of A1, B7, B8, B9, B10 andD6, wherein the variant or derivative is truncated or extended withrespect to any of SEQ ID NO: 14-19 and/or contains one or more aminoacid substitutions, deletions, insertions, and/or additions relative toany of SEQ ID NO: 14-19. Such a targeting moiety retains affinity andselectivity for particular target determinants, such as ICAM-1.Accordingly, targeting moieties as described herein may includepeptidomimetics of any of A1, B7, B8, B9, B10 and D6 their nucleotideencoding sequences, and the viral, bacterial, or cellular systemsexpressing and/or producing these peptides.

Phage-display technology was utilized to identify small 7-mer randomsequence peptides capable of recognizing ICAM-1. Although this techniquehas been proven in the past regarding identification of peptides withrecognition properties, classically phage-display of larger randompeptide sequences is used, (e.g., >12 amino acids) to increase chancesof specific recognition of a determinant. Importantly, althoughtargeting peptides can be generated by this method, whether thesepeptides have the ability to induce (upon binding to their surfacedeterminants) cellular signals to induce transport into and/or acrosscells is totally unpredictable. The importance of this is demonstratedin a recent publication by the present inventors (Garnacho et al., 2008,Journal of Controlled Release, 130:226-233), which indicates thatbinding of targeting moieties to different epitopes or regions of thesame determinant may lead to surface retention of drug carriers, theirendocytic transport, and/or differential intracellular destination, evenwhen the targeted epitopes are in close proximity or even overlapping.Moreover, some targeting moieties do not bind to their targets aftercoupling them to carriers (Garnacho et al., 2008, Journal of ControlledRelease, 130:226-233).

Targeting moieties that are proteins or peptides may be obtained by anymethod known to those of skill in the art. Specifically, such proteinsor peptides may be synthetic or recombinant or may be isolated from anaturally-occurring source or may be identified by phage display.Isolation of proteins or peptides of the invention so identified may beperformed by any known method, including use of oligonucleotides, suchas those described in Example 8 below.

In one embodiment, the targeting moiety is a protein or peptideexpressed from a polynucleotide or expressed from an expression plasmidcontaining a polynucleotide encoding the protein or peptide. Furthermorethe invention includes nucleic acid sequences that encode a targetingmoiety selected from γ3, derivatives of γ3 (e.g., 2γ3 or 3γ3), A1, B7,B8, B9, B10, and D6. The invention also includes nucleic acid sequencesthat encode a peptide of any of SEQ ID NO: 1, 2, 3, 14, 15, 16, 17, 18or 19. In another embodiment, the targeting moiety is a protein orpeptide expressed from a nucleic acid sequence.

Targeting moieties of the invention may be monomeric, dimeric,tetrameric, or any other oligomeric form.

In another embodiment of the invention, the composition comprises morethan one targeting moiety, where the composition comprises a targetingmoiety that is one or more of γ3, 2γ3, and 3γ3, a variant, derivative orpeptidomimetic of γ3, 2γ3, or 3γ3, A1, B7, B8, B9, B10, D6, a variant,derivative or peptidomimetic of A1, B7, B8, B9, B10, or D6, incombination with an antibody, an aptamer, a nucleic acid, a peptide, acarbohydrate, a lipid, a vitamin, a toxin, a component of amicroorganism, a hormone, a receptor ligand and any derivative thereof.

In a further embodiment the invention provides a viral, non-viral,bacterial or cell system containing, encoding, expressing, and/orproducing a targeting moiety. Such systems may also, optionally, furthercontain, encode, express, and/or produce an agent. Nucleic acidsequences encoding one or more targeting moieties and/or one or moreagents may be present on the same nucleotide sequence or may be presenton different nucleotide sequences. Expression of the agent may beco-expression with the targeting moiety or may be separately expressed.

In a still further embodiment, the targeting moiety recognizes aspecific target. Such a specific target may include, but is not limitedto an antigen or a receptor. In one particular embodiment the specifictarget is ICAM-1. In one embodiment of the invention, the targetingmoiety recognizes both a molecular target isoform present in an animalmodel (e.g., mice) and also recognizes the human isoform. Preferably thetargeting moiety demonstrates an affinity for both human and mouseICAM-1.

Binding of the targeting moiety to its target molecule on the cellsurface is contemplated by the invention for tunable drug carrierdelivery and/or transport on the cell surface, intracellularly ortranscellularly, as well as interaction with the cell surface targetproviding secondary protective and/or beneficial effects, e.g., blockageof ICAM-1 binding to leukocytes, fibrinogen/fibrin, and/or pathogens.

In one embodiment the invention provides a delivery composition thatcomprises a targeting moiety that is capable of binding to ICAM-1. In afurther embodiment the targeting moiety binds to human and/or mouseICAM-1.

The binding of the targeting moiety and ICAM-1 or the transfection,expression, or production of the ICAM-1-targeting moiety may occur inany of cell culture, in vivo, ex vivo or in vitro. The binding may occursuch that the targeting moiety binds to and remains on or at the cellsurface, such that the targeting moiety binds to and is internalized bycells, or such that the targeting moiety is transported across thecells. Where the targeting moiety is internalized by the cells ortransported across the cells, such internalization or transport mayprogress by any suitable mechanism, including, but not restricted to,endocytosis mechanisms, such as, but not restricted to, CAM-mediatedendocytosis.

As a part of fibrinogen/fibrin, γ3 is constitutively present in thecirculatory system. Binding of γ3 to ICAM-1 has been shown to decreaseinflammation, it has been postulated that γ3 may attenuateatheroclerosis, cancer, and ICAM-1-dependent infections, and there isevidence that γ3 promotes cell survival and anti-apoptotic effects inactivated endothelial cells. ICAM-1 is involved in and/or contributes toa variety of maladies or disorders. Such involvement or contribution mayinclude interactions such as binding to ICAM-1, ICAM-1 function, ICAM-1signaling, CAM-mediated endocytosis (see below) or, generally,ICAM-1-mediated pathways or pathways including ICAM-1 function (whereICAM-1 contributes either solely or in addition to other molecules).Such maladies or disorders include, but are not limited to,inflammation, immune diseases, thrombosis, oxidative stress, and/orcertain pathogens, such as acute lung injury, acute respiratory distresssyndrome, pulmonary thromboembolism, acute myocardial infarction,ischemic stroke, peripheral vascular pathology, deep vein thrombosis,atherosclerosis, hypertension, diabetes, cancer, AIDS, flu, common cold,poliomyelitis, and malaria.

Accordingly, delivery compositions of the invention, comprising γ3,variants or derivatives thereof (e.g., 2γ3 or 3γ3), A1, B7, B8, B9, B10,and/or D6 are effective in treating such conditions, disorders ormaladies. Specifically, in one embodiment, a delivery composition iseffective to inhibit inflammation. In another embodiment, a deliverycomposition of the invention is effective to inhibit lodging of anembolus or inhibit other occlusion of a blood vessel by lodging in thevasculature. Delivery compositions of the invention may also beeffective in treating conditions such as bacterial infection, cancer andartherogenesis. The Examples as described herein and set forth in detailbelow demonstrate the successful conversion of γ3 and phage-displayderived peptides into targeting molecules to drive binding,intracellular and transcellular transport of therapeutics and theircarriers to cells, and block adhesion of objects to ICAM-1. The Examplesfurther demonstrate the successful inhibition of inflammatorytransmigration of leukocytes across endothelial cells and successful invivo demonstration of inhibition of lodging of fibrin microemboli in thevasculature using compositions of the invention.

As described herein, the invention provides a composition comprising atargeting moiety. In one embodiment the composition is for delivery ofan agent to a cell, where the delivery composition includes both atargeting moiety and one or more agents, where the targeting moietyrecognizes and binds to a target on a cell and is effective to deliverthe agent to the cell. In a further embodiment, the invention provides adelivery composition that includes one or more targeting moieties(recognizing the same or different targets, in the same or differentcells) and one or more agents, where one or more of the targetingmoieties recognizes and binds to a target on a cell, tissue or organ andis effective to deliver the agent to the cell, tissue or organ.

Target locations may include, but are not limited to, targeting of thekidneys, the spleen, the heart, the lungs, the liver or the brain, andtargeting of cells or tissues present in or derived from such organs.The Examples provided herein demonstrate successful in vivo braintransport of therapeutic agent-containing delivery compositions of theinvention.

In one embodiment the invention provides a composition comprising anagent such as, but not limited to, research, analytical or molecularprobes, diagnostic and therapeutic agents, biologically active agents,research agents, analytical agents, imaging agents, monitoring agents,enzymes proteins, hormones, lipids, sugars, nucleic acids, lipoproteins,and chemicals. In another embodiment, where the agent is an enzyme, itcomprises a lysosomal enzyme or encodes for a lysosomal enzyme. Such alysosomal enzyme may be selected from the enzymes involved in PompeDisease, GM1 gangliosidosis, Tay-Sachs disease, GM2 gangliosidosis,Sandhoff disease, Fabry disease, Gaucher disease, metachromaticleukodystrophy, Krabbe disease, Niemann-Pick disease type A,Niemann-Pick disease type B, Niemann-Pick disease type C, Niemann-Pickdisease type D, Farber disease, Wolman disease, Hurler Syndrome, ScheieSyndrome, Hurler-Scheie Syndrome, Hunter Syndrome, Sanfilippo ASyndrome, Sanfilippo B Syndrome, Sanfilippo C Syndrome, Sanfilippo DSyndrome, Morquio A disease, Morquio B disease, Maroteaux-Lamy disease,Sly Syndrome, α-mannosidosis, β-mannosidosis, fucosidosis,aspartylglucosaminuria, sialidosis, mucolipidosis II, mucolipidosis III,mucolipidosis IV, Goldberg Syndrome, Schindler disease, cystinosis,Salla disease, infantile sialic acid storage disease, Batten disease,infantile neuronal ceroid lipofuscinosis, and prosaposin. In oneembodiment of the invention the agent is acid sphingomyelinase.

The composition may further comprise a delivery carrier for thetargeting moiety and/or the agent. In one embodiment, such a deliverycarrier is selected from a natural virus or derived viral-like particle,dendrimer, carbon nanoassembly, liposome, a polymer carrier, amicrobubble, a paramagnetic particle, a ferromagnetic particle, aself-assembled polymer, a polymersome, a filomicelle, a micelle, a microparticle or nanoparticle, an albumin particle, and/or a lipoprotein.

The elements of the composition may be varied, such as by size andshape, to modulate transport of the composition from the plasma membraneinto or across the cells.

In one embodiment the targeting moiety is selected by its size, whereinthe size is optimized for delivery to particular destination(s) within acell. Targeting may be directed to loci such as the lysosome, endosomes,or pathways mediating transport from inside of a target cell to thesurface of the cell. Therefore, in one embodiment, the targeting moietyis either below 1 μm size or larger than 1 μm size.

In one embodiment the composition further comprises sodium protonexchanger 1 (NHE1) inhibitors (such as amiloride) or protein kinase Cactivators (such as phorbol esters) to modulate transport.

Within the composition, the interaction of the elements may occur in amanner providing the most efficient reaction with the target.Accordingly, the interactions may include, but are not limited to, anyof the following: the targeting moiety may be coupled to the deliverycarrier, the agent may be coupled to the targeting moiety, the agent maybe coupled to the delivery carrier, and/or the agent may be coupled toboth the targeting moiety and the delivery carrier. The coupling of theelements of the composition may be any effective means of linking,binding, or conjugating the elements. Such interactions may include, butare not limited to, covalent binding, non-covalent binding, binding as asingle entity, or binding in combination with one or more other elementsof the composition.

The data described herein and reported in the Examples below demonstratethat materials of very different nature and chemistry, size and geometrycan safely and efficiently access both human and mouse ICAM-1 whentargeted by any of γ3, 2γ3, 3γ3, A1, B7, B8, B9, B10, and D6 and deliverviruses, carriers and agents to the cell surface, as well as into andacross cells, via a pathway including ICAM-1 function, operative in avariety of cell types in control and disease conditions.

In one embodiment the invention provides compositions and methods usefulin both laboratory experimentation and clinical endeavors. Thecompositions and methods of the invention are applicable in in vivo, exvivo and in vitro applications, including cell cultures, animal models,human application or administration, and the like.

Comparison was made of γ3, 2γ3, 3γ3, A1, B7, B8, B9, B10, and D6expressed onto viruses or coupled to prototype polystyrene carriers,biodegradable FDA-approved material-PLGA carriers, and carriers bearingtherapeutic enzymes (recombinant acid sphingomyelinase, ASM) vs IgG-,anti-ICAM- and γ3-derived or A1-derived scramble peptide counterpartsand ligands of classical endocytic pathways, and non-targetedcounterparts, in terms of binding, transport, and effects, both in cellcultures and animal models.

In Example 1, in vivo accessibility of ICAM-1 was compared to that ofdeterminants of clathrin and caveolar pathways, which also mediatetransport into and across cells. As an example, antibodies totransferrin receptor and GM1, associated to clathrin pits and caveoli,only gained access to lung endothelium when injected iv in mice as freecounterparts, not on 180 nm carrier particles (FIG. 1A), likely due tosize limits of clathrin pits and caveoli and/or distribution of theirreceptors. ICAM-1 was accessible to both free targeting moieties(antibodies) and preferential carriers, which were specific againstcontrol IgG carriers (FIG. 1A). ICAM-1 targeting increased in a diseasemouse model (ASM knockout (KO) mice), likely due to ICAM-1overexpression described in many pathologies, while accumulation of freeASM, an enzyme that binds to mannose-6-phosphate receptor associated toclassical clathrin pathways, was 10-fold lower and was further decreasedin the disease model (FIG. 1B). This could be due to reduced endocyticuptake via classical pathways in disease conditions, as Niemann-PickA/B-patient cells and ASMKO-mice cells had reduced uptake of ligands,toxins, and particles by clathrin pits, caveoli, macropinocytosis, andphagocytosis (FIG. 1C). Endocytic defects were also observed in otherdiseases, including Fabry, Gaucher and Niemann-Pick C (FIG. 1D). Yet,CAM-endocytosis of ICAM-1-targeted carriers was as efficient as incontrol cells (FIG. 1C). Also, ICAM-1-targeted particles of varioussizes (up to 5 μm) and shapes (spheres, elliptical disks, andpolymorphous conjugates) could access ICAM-1 targets in vivo and beefficiently endocytosed in cell culture (FIG. 1E).

In Example 2, ICAM-1-targeting moieties were shown to stably target thecell surface (anti-ICAM in FIG. 2A) and intracellular compartments(anti-ICAM polystyrene nanocarriers in FIG. 2B) in cell culture, as wellas in vivo after iv injection in mice (anti-ICAM polystyrene carriers inendosomes and lysosomes in FIG. 2C left), where they were also safelytransported across endothelial cells layers transcellularly withoutdisruption of the cell junctions (FIG. 2C right). The final destinationof these carriers, to compartments within the cell or re-surfacing tothe exterior of the cell (adequate for intracellular vs transcellulardelivery) can be controlled by the size of the carriers (FIG. 2D).

Example 3 demonstrates broad utility of ICAM-1-mediated targeting andtransport for a variety of drug delivery systems and cell types.ICAM-1-targeted systems were tested, including biotin-streptavidinconjugates, polystyrene particles, PLGA carriers, poly-ethylene glycolpoly-lactic acid (PEG-PLA) carriers, and natural polymer dendrimers,were efficiently internalized by cells in culture (FIG. 3A); and alltested endothelial cells, such as lung and brain endothelium, frommacro- and micro-vascular beads, of mouse and human origin (FIG. 3B),and also by non-endothelial cells, including macrophages, alveolarepithelial cells, and neuroblastoma cells (FIG. 3C). Hence, altogetherthis series of experiments demonstrate the efficacy and versatility orICAM-1 targeting in the context of delivery of drug carriers in cellcultures and in vivo, to different sub-cellular environments, in controland diseased conditions.

Example 4 demonstrates efficient and specific binding and intracellulartransport of therapeutic carriers to human and mouse ICAM-1 via theaffinity peptide γ3 and its derivative peptides 2γ3 and 3γ3.Furthermore, synthetic peptide γ3 was absorbed on 100 nm FITCpolystyrene particles and the resulting carriers efficiently bound toimmobilized human ICAM-1/Fc (as expected) but also, surprisingly, tomouse ICAM-1/Fc, vs to control immobilized albumin (FIG. 4A). Bindingwas similar to that of anti-ICAM carriers (FIG. 4A). Hence, γ3 can beused in mouse and human models and settings. The γ3 carriers also boundto native ICAM-1 expressed by both activated human and mouse endothelialcells, but not control 293 cells which are known to be voided of ICAM-1expression (FIG. 4B). Importantly, co-absorption of the therapeuticenzyme ASM with γ3 on the carrier surface did not affect targeting tohuman or mouse endothelial cells (FIG. 4B). Targeting to these cells wassimilarly suppressed by excess of free γ3 peptide or anti-ICAM in themedia, but not by a peptide with a scrambled γ3 sequence (FIG. 4B).Excess free mannose-6-phosphate (MP6) in the media did not compete forcarrier binding to cells, either, indicating that M6P residues in ASMdid not contribute to targeting (FIG. 4B).

The γ3/ASM polystyrene carriers were internalized by human and mouseendothelial cells at 37° C., and after uptake, γ3/ASM carrierstrafficked to lysosomes in the perinuclear region of the cell, shown byco-localization with dextran-labeled lysosomes in HUVEC (FIG. 4C).Binding and internalization leading to lysosomal transport was similarfor carriers targeted to ICAM-1 by either γ3 or their derivatives 2γ3and 3γ3 (FIG. 4D). Endocytic transport was inhibited by amiloride andactivated by phorbol esters activating protein kinase C or PKC,confirming a CAM-mediated mechanism (FIG. 4E). Hence, γ3 carriers elicitthe same unique pathway than anti-ICAM carriers, crucial to effectivelydeliver therapeutics intracellularly and across cells.

Example 5 details development of the 7-mer phage-display derivedpeptides against ICAM-1. These were identified and isolated from arandom library by using a chimeric ICAM-1 protein consisting of the twomost membrane-distal Ig domains of mouse ICAM-1 fused to human Fc (hFc).One round of negative selection against hFc, followed by three rounds ofpositive selection against mouse ICAM-1/hFc, rendered six phage clones(A1, B7, B8, B9, B10, and D6), which recognized immobilized mouse ICAM-1(FIG. 5A). Specificity vs hFc was higher for A1 (˜5.1 fold), followed byB8>B7>B9>B10>D6 (68.6%-41.2% of A1) (FIG. 5B). A1 clone presented thehighest affinity to immobilized mouse ICAM-1 (˜4.7 fold overnon-selected phages), followed by B7>B8>D6>B9>B10 clones (85.7%-56.7% ofA1) (FIG. 5C). All six clones recognized native ICAM-1 expressed bymouse endothelial cells (H5V) as compared to control 293-ICAM-1negative-cell type. In this setting, also A1 phage clone presented thehighest affinity followed by D6>B10>B7>B8>B9 clones (92.9%-72.3% of A1)(FIG. 5D). Binding to cells increased up to 1.8 fold by TNFα, a cytokinewhich up-regulates ICAM-1 expression (FIG. 5D).

In addition, fluorescence microscopy showed that phage virusesexpressing 5 copies of the peptides A1, B7, B9, or D6 (but not B8 orB10) on their capsid were internalized by endothelial cells at 37° C.(65-87% internalization, 1 h) but not at 4° C., indicative ofendocytosis (FIG. 5E). This process was inhibited by amiloride (41-59%inhibition) (FIG. 5F), which has been shown in the past to interferewith CAM-mediated endocytosis. Therefore, these peptides are alsoadequate for either stable delivery to the cell surface, orintracellular delivery.

Surprisingly, clone A1 phages also strongly recognized immobilized humanICAM-1/hFc and native human ICAM-1 expressed by human endothelial cells(HUVEC), (˜70% affinity of mouse ICAM-1). A1 peptide synthesized invitro (but not control A1 scramble peptide), as well as B9 (and at someextent B8 and B9) competed binding of anti-ICAM nanocarriers to humanendothelial cells (˜80% inhibition) (FIG. 5G). This is a crucialfinding, since it provides the opportunity to utilize these peptides inmouse and human cells and settings, and potentially in future clinicaltrials. Also, this demonstrates that ICAM-1-targeting peptides can blockbinding of other objects to ICAM-1, e.g., for applications such asbeneficial blockage of binding of leukocytes, fibrinogen/fibrin, and/orpathogens to ICAM-1.

Example 6 demonstrates in vivo pharmacokinetics and biodistribution oftherapeutic carriers targeted to ICAM-1 via the affinity peptide γ3 andits derivative peptides 2γ3 and 3γ3. In animal model tests, the γ3 2γ3or 3γ3 carriers, coupled to therapeutic ASM, bound specifically andefficiently to, and were endocytosed by, endothelium in vivo in mice.They rapidly disappeared from the bloodstream (FIG. 6A), which was not aconsequence of rapid clearance by the reticuloendothelial system (as inthe case of clearance of control IgG/ASM carriers by the liver) butrather a consequence of rapid targeting to organs, e.g., the lungs,after i.v. injection in mice (FIG. 6B). Indeed, γ3 2γ3 or 3γ3 carriersenhanced the delivery of ASM vs than of free ASM in all organs,including brain, kidneys, spleen, liver, heart, and lungs, and 2γ3 or3γ3 carriers showed lower lung deposition and greater brain depositionthan γ3 carriers (specificity index, FIG. 6C) This is the first timethat test brain targeting of ICAM-1 strategy has been examined andsurprisingly its efficacy was found to be higher than the one reportedfor strategies of targeting to determinants commonly used for braindelivery, such as the case of insulin receptor or transferrin receptor:targeting to insulin receptor of α-L-iduronidase=0.02% injected dose pergram (% ID/g) versus ICAM-1 targeting of ASM prototype carriers=0.2%ID/g in brain.

Accumulation of ASM delivered by γ3, 2γ3 or 3γ3 carriers to these organswas comparable to that of ASM delivered by anti-ICAM carriers (FIG. 6D).This is readily clear from the localization ratio of these formulations,which represents the ratio between the % injected dose per gram of organand that in circulation. Since the estimated dose measured in a givenorgan also include that of the blood irrigating that organ, thelocalization ratio permits to normalize data to the different levels ofcirculating materials. For instance, ASM lacks affinity and results inhigh circulating levels compared to ICAM-1 targeted formulations thatrapidly bind to endothelium and disappear from blood. ASM targeting invivo to ICAM-1 by the γ3-strategy was specific, as shown by lack oftargeting of 2γ3-scramble/ASM carriers, or that of 2γ3/ASM carriers inICAM-1 KO mice (FIG. 6E). Importantly, targeting to ICAM-1 withbiodegradable PLGA carriers by the γ3-strategy resulted in CAM-mediatedendocytic transport in vesicular compartments in endothelium in vivo(e.g., 2γ3/ASM PLGA carriers in FIG. 6F).

Example 7 demonstrates the therapeutic effects of cargoesintracellularly delivered to patient cells by γ3-strategy carriers(Table 1).

As an example of the therapeutic utility of this strategy, the effectsof ASM targeting were compared to ICAM-1 using anti-ICAM vs theγ3-strategy provided herein. Recombinant ASM is used for enzymereplacement therapy of patients of types A and B Niemann-Pick disease,where genetic mutations lead to ASM deficiency and intracellularlysosomal accumulation of sphingomyelin and cholesterol, resulting incell dysfunction, multi-organ complications, and often a fatal outcome.(He X., et al., Anal. Biochem. 2003; 314:116-20; Schuchman E. H., andMuro, S., The development of enzyme replacement therapy for lysosomaldiseases: Gaucher disease and beyond. In: Futerman A H, ed. Gaucherdisease: Lessons learned about therapy of lysosomal disorders: CRCPress.; 2006:125-40.) It has been previously shown by the presentinventors that anti-ICAM carriers deliver active ASM to lysosomes incells from Niemann-Pick patients, attenuating sphingomyelin andcholesterol storage to control values, vs free ASM which only partiallyattenuates the levels of these lipids. Similar levels of degradation ofthese lipids were achieved by targeting ASM to ICAM-1 via the present γ3strategy, using either prototype polystyrene carriers or biodegradablePLGA carriers (Table 1).

Example 8 provides a strategy for the design of a chimeric therapeuticenzyme containing ICAM-1-targeting γ3, 2γ3 or 3γ3 derivative peptides,or A1, B7, B8, B9, B10, or Dl peptides.

Further, direct coupling of γ3, 2γ3, 3γ3, A1, B7, B8, B9, B10, and/or D6peptides (or their nucleotide encoding sequences) to therapeutic ordiagnostic agents, via biotin-streptavidin, chemical conjugation,covalent coupling, through antibodies, or direct synthesis (e.g.,chimeric proteins or their coding sequence for gene expression viaplasmid, viral or non-viral vectors), is contemplated for utility forICAM-1 targeting and transport to cells or the body in vivo. As anexample, FIG. 7 shows the design of a therapeutic chimeric enzymeproduced from a plasmid containing γ3, 2γ3 or 3γ3 sequences cloned atthe amino-terminus of the ASM sequence, separated by a (Ser4-Gly)2peptide spacer to allow independent folding and the targeting andenzymatic moieties of the chimera. This cassette can be cloned in apMT/BiP/V5-His (B) plasmid for amplification under ampicillin selectionin E. coli and expression in S2 insect eukaryotic cells, which willallow for expression under metallothionein promoter upon induction bycopper sulfate, traffic of the chimeric enzyme through the secretorypathway due to presence of BiP, and extracellular secretion after BiPcleavage by S2 cells. The chimeric enzyme, which contains V5 sequenceand 6His tag fused to the carboxyl terminus, can be purified using aNi-chelating resin. The resulting protein (˜80 kDa) can be separated bySDS-PAGE and blotted with anti-V5 to trace the V5-tag. Possiblemodifications of this design include elimination of the BiP, V5 and/orHis-tag sequences, elimination of change of the linker, cloning of thetargeting peptide in the carboxyl-terminus of ASM, exchange of the ASMcoding sequence for another therapeutic gene, siRNA, or cloning of thetargeting peptide with no cargo, tandem repeats of the targeting peptideto allow for multivalency of ICAM-1 targeting, inclusion of interactingpeptides or sequences to promote dimerization, tetramerization, orformation of oligomers of the peptides or the chimera, and to providemultivalent targeting to ICAM-1, cloning into other vectors forexpression under different selection markers, in different cell types,in bacteria, by viruses, for enzyme or gene therapy, among othermodifications.

Example 9 provides in vivo brain transport of a composition of theinvention in a mouse model, where the composition contains 2γ3/ASM andPLGA. FIG. 9 is transmission electron microscopy pictures showingsuccessful migration of the compositions across the blood-brain barrier.

Example 10 provides inhibition of inflammatory leukocyte transmigrationacross endothelial cells by ICAM-1-targeting peptides A1, B7, B8, B9mB10 and D6. All were shown (FIG. 9) to inhibit leukocyte transmigration,when compared to the controls (albumin (negative control), scrambledpeptide (negative control) and LB2 (positive control)).

Example 11 provides in vivo inhibition of lodging of fibrin microemboliin the vasculature of mice by administration of ICAM-1-targeting peptideγ3, resulting in continued circulation of the fibrin microemboli, ratherthan lodging in the vasculature or other organ.

Therefore, it is possible to safely and effectively target therapeuticsand their carriers (including viruses) to both human and mouse ICAM-1,in a variety of cell types in culture and in vivo in animals and animalmodels, using the γ3 peptide, derivative peptides 2γ3 or 3γ3, and/orphage-derived peptides A1, B7, B8, B9, B10, or D6. This provides eitherstable binding to the cell surface and/or further transport tointracellular compartments and/or across cells for penetration in thetissues, with delivery of active compounds providing therapeuticbenefits, as in the case of enzyme therapies and/or gene therapies forlysosomal disorders (see U.S. Provisional Patent Application60/584,648). Exemplary carriers for safe transport of materials fromintracellular compartments such as endosomes into the cell cytosol andnucleus are described in U.S. Provisional Patent Application 60/931,552.The present ICAM-1-targeting strategy in combination with these carriershave general applicability for delivery to these cell compartments.

ICAM-1 serves as an adhesive surface for leukocytes during inflammation(L. Yang, et al., Blood 106, 2 (2005): 584-92; S. Muro, VCAM-1 andICAM-1. In: Aird W. (Ed), The Endothelium: A Comprehensive Reference.Cambridge Univ. Press. 2007: New York. 1058-1070; Rothlein R, et al., JImmunol 1986; 137:1270-4.). It is constitutively expressed on diversecell types, including (but not restricted to) endothelial, Schwann,glial, and epithelial cells, leukocytes, myocytes, etc (S. Muro, VCAM-1and ICAM-1. In: Aird W. (Ed), The Endothelium: A ComprehensiveReference. Cambridge Univ. Press. 2007: New York. 1058-1070; Hopkins, A.M., et al. Adv Drug Deliv Rev 56 (2004): 763-778.) Since ICAM-1 isexpressed in diverse cell types in the body, including epithelial cellslining most of body cavities and entry routes (e.g., epithelial cells inthe airways and alveoli, epithelial cells in the gastrointestinal tract,mesothelial cells in the pleura, and epithelial cells lining joints andventricles in the CNS), muscle cell, glial and neuronal cells, etc., thecompositions and methods described herein are useful for delivery ofdrugs to all of these compartments in the body. Furthermore, delivery islikely to be enhanced under pathological conditions due tooverexpression of ICAM-1 and ICAM-1 blocking may be additionallybeneficial (anti-inflammatory effects, anti-thrombotic by preventingdeposition of fibrinogen and/or fibrin). Similar methods could beapplied for intracellular delivery of drugs, biosensors, research,analytical, imaging, diagnostic and therapeutic agents, either in thecontext of research or clinical applications.

ICAM-1 is an attractive target to achieve selectivity toward diseasesites in the body, since the expression of this molecule is up-regulatedin many pathologies (S. Muro, VCAM-1 and ICAM-1. In: Aird W. (Ed), TheEndothelium: A Comprehensive Reference. Cambridge Univ. Press. 2007: NewYork. 1058-1070; Rothlein R, et al., J Immunol 1986; 137:1270-4;Hopkins, A. M., et al. Adv Drug Deliv Rev 56 (2004): 763-778; Hubbard,A. K. & Rothlein, R. Free Radic Biol Med. 28 (2000): 1379-1386.).Targeting ICAM-1 does not seem to lead to side effects and indeed thisstrategy is being explored to block of ICAM-1 adhesive function andprovide side benefits, such as anti-inflammatory effects (Takei, Y. etal., Transplant Proc 28 (1996): 1103-1105; Kavanaugh, A. F., et al.,Arthritis Rheum 40 (1997): 849-853; Hallahan, D. E. & Virudachalam, S.PNAS. 94 (1997): 6432-6437.). For instance, antibodies to ICAM-1 arebeing explored as therapeutics and affinity carriers in cell cultures,animal models, and early clinical studies, where they have shown goodsafety (Muro, S. et al., Mol Ther. 13, 1 (2006): 135-141; Garnacho, C.et. al. JPET 325 (2008): 400-408; Muro, S. et al., Molecular Therapy 16,8 (2008): 1450-1458; Murciano, J. C., et al., Blood 101 (2003):3977-3984; Villanueva, F. S., et al., Circulation 98 (1998): 1-5;Weller, G. E., et al., Ann Biomed Eng. 30 (2002): 1012-1019; Danilov, S.M. et al., Am J Physiol 280 (2001): L1335-L1347; Sakhalkar, H. S., etal., Proc Natl Acad Sci USA 100 (2003): 15895-15900; Rossin, R., et al.,J. Nucl. Med. 49, 1 (2008): 103-111; Muro, S., et al., Blood. 105(2005): 650-658.). The short peptides and peptides identified byphage-display described herein also serve as ICAM-1 targeting moleculesto provide efficient and specific binding of therapeutic agents and drugdelivery systems to ICAM-1 in both mice and humans.

Coupling of multiple ICAM-1-targeting moieties (e.g., anti-ICAMantibodies) on the surface of carriers has been shown to provide bindingof carriers to ICAM-1 expressed on the plasma membrane of cells (Muro,S., et al., Am J Phys-Cell Physiology. 285, 5 (2003): C1339-47.).Carrier binding through multiple ICAM-1-targeting molecules (multivalentbinding) causes ICAM-1 to cluster, which initiates signal transductionpathways leading to uptake of such carriers into cells by an endocyticmechanism known to as Cell Adhesion Molecule- or CAM-mediatedendocytosis, which is distinct from classical mechanisms ofmacropinocytosis, phagocytosis, clathrin- or caveolar-mediatedendocytosis described above (Muro, S., et al., Am J Phys-CellPhysiology. 285, 5 (2003): C1339-47.). However, when presented to cellsas non-coupled counterparts (in contrast to multivalent binding) bindingof the ICAM-1-targeting moieties (e.g., anti-ICAM) to ICAM-1 onendothelial cells does not elicit this endocytic transport mechanism.This precludes the use of ICAM-1-targeting molecules per se (without acarrier scaffold or another mechanism to provide multivalent binding toICAM-1) from being used for drug delivery into or across cells.

In addition, CAM-mediated endocytosis has only been demonstrated inendothelial cells that constitute the inner layer of the blood vesselbut not in epithelial cells from GI tract. Endothelial cells arebelieved to have a very active endocytic capacity given that they usethis mechanism of transport to control exchange of large macromoleculesand blood cells between the blood and the tissues, whereas GI epithelialcells transport most substances via cell junctions, non-endocyticchannels and carrier proteins in their membrane, which are not suitablefor safe transport of large bulky drug carriers (Owen, R. L. Semin.Immunol. 11 (1999): 157-163; Hidalgo, I. J. & Borchardt, R. T., Biochim.Biophys. Acta 1028 (1990): 25-30; Muro, S., et al. Curr. Vasc. Pharm. 2(2004): 281-299; Predescu, D., et al., Am. J. Physiol. Lung Cell Mol.Physiol. 287 (2004): L895-L901.). It was therefore uncertain whether thesignal cascades and other cell machinery related to CAM-mediatedendocytosis is in place in GI epithelial cells and whether suchmechanisms could be employed in transport of carriers across theepithelial tract.

Furthermore, polarized epithelial cells of the GI tract, with multipleprotruding microvilli in the apical membrane in contact with the GIlumen, have a morphology categorically different from the flat easilyaccessible apical surface of endothelial cells in blood vessels, whichcan pose a tremendous accessibility obstacle to ICAM-1-targetingmoieties and/or carriers. Moreover, endothelial cells are believed toexpress the highest level of ICAM-1, which may contribute to inductionof CAM-mediated endocytosis, whereas ICAM-1 levels in other cells areonly relevant or have increased relevance by up-regulation duringpathological stimuli but are not relevant or have decreased relevanceunder normal conditions, which is likely to be the case for delivery ofdrugs needed in distal diseased areas across a healthy GI tract (Muro,S., et al. Curr. Vasc. Pharm. 2 (2004): 281-299; Rothlein R, et al., JImmunol 1986; 137:1270-4.). Finally, all characterized ICAM-targeteddelivery systems underwent intracellular trafficking via endosomalvesicles to either plasma membrane recycling pathways (in rareinstances) or (in most instances) to lysosomal delivery and degradation(Muro, S., et al., J Cell Sci, 116, 8 (2003): 1599-1609; Muro, S. &Muzykantov, V. R., Curr Pharm Des. 11 (2005): 2383-2401; Muro, S. etal., Mol Ther. 13, 1 (2006): 135-141.) but not to cross epitheliallayers by transcytosis. In fact, ICAM-1 serves as an adhesion surfacefor leukocytes during inflammation and is involved in the process ofleukocyte migration into tissues by opening of cell junctions linked tothe paracellular transport (Muro, S., et al. Curr. Vasc. Pharm. 2(2004): 281-299; Rothlein R, et al., J Immunol 1986; 137:1270-4; L.Yang, et al., Blood 106, 2 (2005): 584-92.), which is opposite to safetransport by the transcellular route.

In consideration of these known characteristics of ICAM and GIepithelial cells, the possibility of ICAM-1 targeting to and transportacross healthy GI epithelial cells by a transcellular mechanism wasunlikely, uncertain and unpredictable. As set forth in the examplesbelow, the present inventors have demonstrated methods of transport anddelivery employing CAM-mediated endocytosis in the GI epithelial tract.

The examples provided herein demonstrate that GI epithelial cellssupport: (i) efficient and specific targeting of ICAM-1-targetedcarriers both as non-activated healthy cells and also as pathologicallyaltered cells, as well as (ii) safe, fast, and efficient transport ofsuch carriers across their cellular body in both healthy andpathological conditions, via CAM-mediated endocytosis with no damagingopening of cell junctions. GI epithelial cells also supported targetingand CAM-mediated endocytosis of non-multivalent, non-coupledICAM-1-targeting moieties (anti-ICAM) in the absence of carrierparticles.

In the examples it was shown that GI epithelial cells express ICAM-1 andthat anti-ICAM carriers bind to such GI epithelial cells efficiently andthat uptake of the anti-ICAM carriers by GI epithelial cells isCAM-mediated endocytosis (Examples 12-15). A model representative of theGI epithelium was generated (Example 16) and tested for ICAM-1expression (Example 17) and rapid anti-ICAM carrier transport (Examples18 and 19). It was further shown that CAM-mediated endocytosis isinvolved in the transcytosis of anti-ICAM carriers in the model (Example20), even in the case of non-multivalent binding to ICAM-1 (Example 21).

The examples therefore demonstrate that: (i) ICAM-1-targeted carriersbind efficiently to GI epithelial cells, such binding is specific toICAM-1, and binding is relatively fast; (ii) ICAM-1-targeted carriersare transported across GI epithelial cells in a relatively efficient andfast manner; and (iii) transport of ICAM-1-targeted carriers does notoccur via the paracellular pathway that disturbs the epithelialpermeability barrier, but rather it occurs by an endocytic vesicularpathway related to CAM-mediated endocytosis.

The present invention therefore further provides specific strategies foreffective targeting of GI epithelial cells, providing fast, safe andeffective transport across the epithelial layer with no effect on the GIpermeability and which provides increased bioavailability of the agent.In a particular embodiment, ICAM-1, expressed on the surface of GIepithelial cells is targeted.

In a preferred embodiment, the administration of the composition fordelivery across the GI epithelium is provided to the subject orally.However, administration may be carried out in any manner sufficient toprovide the composition to the digestive tract and/or in contact withthe gastrointestinal epithelium.

Therefore in one embodiment the invention provides a compositioncomprising a targeting moiety comprising an anti-ICAM antibody; and anagent, wherein the targeting moiety recognizes and binds to ICAM-1 on aGI epithelial cell and the composition is transported across the GIepithelium. In a particular embodiment the composition is orallyadministered to a subject.

As previously noted herein, compositions of the invention may furthercomprise a delivery carrier. In one embodiment the delivery carrier iseffective to transport the targeting moiety and the agent of thecomposition to the GI epithelial cell for uptake and/or transport.

Compositions of the invention may further comprise a protective agent.In a particular embodiment the protective agent is effective to protectthe targeting moiety and the agent from degradation prior to bindingICAM-1. Polymers and materials can be used to provide protection toICAM-1-targeted carriers against potential degradation in the stomachwhile in transit to the intestine (Jung, T., et al., Euro. Journ. Pharm.and Biopharm. 50 (2000): 147-160), such as, but not limited to, polymerscontaining amino groups providing an acidic pKa, so that the polymer isinsoluble at neutral pH and can be formulated as a tablet-like matrix orhydrogel containing carriers embedded in its structure, to protect themfrom rapid degradation in the GI. As such polymers pass through the acidpH in the stomach, their amines can become protonated and positivelycharged, allowing the polymer to transition to a soluble state. Gradualprotonation of the polymer would favor bioadhesion to the negativelycharged epithelial mucosa, and gradual dissolution would releaseICAM-1-targeted carriers in situ, which will induce ICAM-1-mediatedendocytic transepithelial transport with carrier release into thecirculation. FIGS. 21A and 21B illustrate such exemplary use of aprotective agent, where FIG. 21A illustrates protection of anti-ICAMcarriers from degradation in the stomach by polymers (e.g., chitosan),FIG. 21B illustrates dissolution of polymer gels and gradual carrierrelease after passage through the stomach, and FIG. 21C illustratesbinding and endocytic transcellular transport (vs paracellulartransport) of carriers across the GI epithelial layer. A fraction of thecarriers may also be delivered to lysosomes, where carriers may alsorelease therapeutics, e.g., in the case of treatment of lysosomalstorage disorders using recombinant enzymes, such as the case ofdeficiency of acid sphingomyelinase (ASM) in Niemann-Pick disease.

Protection of the targeting moiety and the agent may further compriseprotection from the immune system of the subject. One limitation of GIdrug delivery which could impede transport is the mucosa-associatedlymphoid tissue (MALT), which contains various immune cells and maydestroy some carriers, and M cells that sample antigens from the GIlumen, which are then delivered to the underlying MALT. In order toevade detection by immune cells of the gut, as shown in FIG. 22,carriers can be coated with certain polymers, including, but not limitedto, polyethylene glycol (PEG), which acts by masking antigens (Moghimiet al., 2003) (e.g. anti-ICAM or ICAM-1-targeting peptides). To stillachieve targeting, ICAM-1-targeting moieties can be attached to carriersvia the PEG spacer arm, so that the targeting moiety is extended outsideof the dense PEG brush for binding to ICAM-1. Substitution of anti-ICAMby small ICAM-1 targeting peptides as described herein may also providean avenue for optimization of this strategy. Therapeutic and/ordiagnostic agents can be carried within the interior of the carrier, onits surface, or coupled to PEG chains (not limited to the describedlocations).

Additionally, since ICAM-1 is also expressed in endothelial and othercell types it is likely that upon transport of ICAM-1-targeted carriersand their cargoes across GI epithelial cells and into the circulation,the compositions will subsequently target the endothelium and tissuesthat express ICAM-1. Because ICAM-1 expression is up-regulated underpathological conditions, upon transport into the circulation, suchstrategy will provide preferential targeting and delivery todisease-affected areas.

In addition, given that pathologically altered GI epithelial cells(e.g., human epithelial colorectal adenocarcinoma cells, TNFα treatedcells which are a model of GI inflammation, etc), it is likely that thisstrategy may be also used to treat pathologies pertinent to the gut,such as colorectal carcinoma, inflammatory bowel disease, Crohn'sdisease, and ulcerative colitis, and bacterial infections (Dippold, W.,et al., Gut. 34 (1993): 1593-1597; Kelly, C. P., et al. Am J PhysiolGastrointest Liver Physiol 263 (1992): G864-G870; Huang, G., et al., J.Clinical Investigation. 98 (1996): 572-583). The ability to effectivelyaccess these intestinal maladies promotes the possibilities that existfor clinical applications involving ICAM-1 targeting.

In a further embodiment, compositions of the invention may comprise asecond or additional targeting moiety. Such targeting moiety may beeffective to target a cell, tissue or organ after transport across thegastrointestinal epithelium and into the circulatory system, such thatthe second targeting moiety recognizes and binds to a target on thecell, tissue or organ, and is effective to deliver the agent to thecell, tissue or organ.

In a still further embodiment, the invention provides methods of usingthe compositions described herein. In one embodiment the inventionprovides a method for delivery of an agent across the gastrointestinalepithelium, comprising administration of a composition comprising atargeting moiety selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, and an anti-ICAM antibody; and an agent, wherein thetargeting moiety recognizes and binds to a target on a gastrointestinalepithelial cell and the composition is transported across thegastrointestinal epithelium. In a particular embodiment the compositionis orally administered to a subject. In a further embodiment the targetis a cell adhesion molecule (CAM) expressed on the surface of thegastrointestinal epithelial cell, e.g., ICAM-1.

As described herein, the preferred uptake and transport of thecomposition across the epithelium is a transcellular route. Such a routeis advantageous in that it does not cause disruption of the permeabilitybarrier. The compositions and methods of the invention provide suchtransport.

The compositions and methods of the invention therefore demonstratesignificant therapeutic benefits of ICAM-1-targeted carriers in GI andoral drug delivery.

The advantages and features of the invention are further illustratedwith reference to the following examples, which are not to be construedas in any way limiting the scope of the invention, but rather asillustrative of various embodiments of the invention in specificapplications thereof.

Example 1 Efficacy and Versatility of Targeting to and TransportMediated by ICAM-1 Vs Determinants of Classical Endocytic Pathways

(A) Lung uptake (30 min) of ¹²⁵I-labeled anti-transferrin receptor(targeted to the clathrin route), anti-GM1 (anti-ganglioside GM1,targeted to caveoli), or anti-ICAM, injected intravenously in mice asfree antibodies or absorbed on the surface of 100 nm polystyreneparticles (prototype carrier final size=180 nm). Particles had¹²⁵I-labeled IgG as a tracer. Dashed line=control unspecific IgGcarriers. (B) Lung uptake (30 min) of radiolabeled acidsphingomyelinase, a recombinant enzyme which binds to mannose 6phosphate (M6P) receptor, vs anti-ICAM polystyrene carriers in controlC57Bl/6 mice vs diseased ASMKO mice. Data is normalized per gram due tolarger lung size in ASMKO mice. Dashed line=anti-ICAM carriers injectedin control ICAM-1 KO mice, which do not express ICAM-1. (C) Endocytosis(1 h-37° C.) of fluorescent transferrin (targeted to the clathrinroute), cholera toxin B (targeted to the caveolar route), EGF-induceddextran (macropinocytosis), and 180 nm anti-ICAM polystyrene carriers(CAM-endocytosis) by fibroblasts from patients of type A-B Niemann-Pick,or 1 μm IgG-coated particles (phagocytosis) by diseased ASMKO alveolarmacrophages, analyzed by fluorescence microscopy. (D) Internalization (1h-37° C.) of fluid-phase Texas-red dextran by skin fibroblast fromFabry, Gaucher, type C Niemann-Pick (NPC) and NPD patients,characterized by deficiency of α-galactosidase-A, glucocerebrosidase,NPC1 transporter, and ASM, respectively. (E) FITC-labeled anti-ICAMcarriers of diverse geometry (anti-ICAM polystyrene spheres from 0.1 to5 μm or 0.1×1×3 μm elliptical disks, and polymorphous anti-ICAMconjugates formed by biotin-streptavidin crosslinking were incubatedwith TNFα activated human umbilical vein endothelial cells (HUVEC) for 1h at 37° C. The cells were then washed and fixed, cell surface-locatedcarriers were differentially stained with a texas red-conjugatedsecondary antibody, and the samples were analyzed by fluorescencemicroscopy to quantify the percent of carriers internalized by thecells. Data are mean±standard error of the mean for n≧4 mice or ≧20cells. *p<0.05, **p<0.005, ***p<0.001 by student's t test.

Example 2 Transport to Different Sub-Cellular Environments Via ICAM-1Targeting

(A) TNFα activated HUVEC were incubated for 1 h at 37° C. with anti-ICAMantibody, then non-bound antibodies were washed and cells were stainedwith a secondary antibody conjugates to texas red, only accessible tocell surface-located anti-ICAM, to detect the antibody fraction thatremained in the plasma membrane. Then, cells were permeabilized andstained with a secondary antibody conjugated with green FITC, whichwould be accessible to both surface and internalized anti-ICAM. Afteranalysis by fluorescence microscopy, cell surface antibodies werevisualized both under the green and red channels, indicated by yellowishfluorescence, whereas no green internalized antibodies were observed.(B) HUVEC were incubated for 1 h at 37° C. with anti-ICAM antibodiesabsorbed on the surface of 100 nm FITC-labeled polystyrene nanocarriers.Cells were then washed, fixed, and stained with texas red secondaryantibody which can only detect accessible surface anti-ICAM carriers.After analysis by fluorescence microscopy, internalized carriers werevisualized under the green channel in the perinuclear region of thecell, whereas no yellowish carriers on the cell surface were observed.Dashed lines in (A) and (B) mark the cell border, determined byphase-contrast microscopy. (C) Anti-ICAM/ASM polystyrene carriers wereinjected in anesthetized C57Bl/6 mice. Five hours after injection, theanimals were perfused and fixed through the left ventricle underventilation, and the lungs were isolated and processed for transmissionelectron microscopy. Arrows=endothelial cell (EC) surface-boundcarriers. White arrowhead=carriers in an intracellular endosome. Blackarrowheads=carriers in intracellular lysosomes. Grey arrowhead=intactcell junctions. Asterisks=caveoli. Bordered arrowheads=carrierstransported across the endothelial layer into epithelial cells in thealveoli. Scale bar=100 nm. (D) Activated HUVEC were incubated for 30 minat 4° C. with FITC-labeled anti-ICAM polystyrene carriers (spherical 180nm or 1 μm, or discoidal 0.1×1×3 μm) to only allow binding, following bywashing and endocytosis of pre-bound particles for 1 h (not shown) or 5h at 37° C. The cells were then fixed and the presence of particlescontaining intact anti-ICAM coat on the cell surface was assessed byimmunostaining using a Texas-red secondary antibody. Since after 1 hincubation no red+green (yellow) particles were visualized in the cellsurface, the appearance of such particles at later times indicaterecycling from intracellular compartments, quantified by fluorescencemicrocopy. *p<0.05, by student's t test.

Example 3 Broad Utility of ICAM-1-Mediated Targeting and Transport for aVariety of Drug Delivery Systems and Cell Types

(A) TNFα activated HUVEC were incubated for 1 h at 37° C. with anti-ICAMconjugates prepared by coupling via streptavidin biotinylated anti-ICAM,anti-ICAM polystyrene nanocarriers, anti-ICAM poly-lactic co-glycolicacid (PLGA) nanocarriers, anti-ICAM poly-ethylene glycol (PEG)poly-lactic acid (PLA) nanocarriers, or biopolymeric dendrimers.Non-bound conjugates and carriers were washed, the cells were fixed,stained and analyzed as described in FIG. 2 A-B. (B) Internalization of100 nm FITC-labeled (green) anti-ICAM polystyrene carriers byTNFα-activated macrovascular human umbilical vein endothelial cells(HUVEC), mouse pulmonary microvascular endothelial cells (PMVEC), humanbrain microvascular endothelial cells (BMVEC), and (C) mouse peritonealmacrophages, human alveolar epithelium-derived EAhy926, and humanneuroblastoma SH-SY5Y. In all pictures cell surface-bound materials areshown in red+green double labeled color (yellowish) vs internalizedmaterials which appear as single labeled in the green channel. Dashedlines mark the cell border, determined by phase-contrast microscopy.Magnification bar=10 μm.

Example 4 Efficient and Specific Binding and Intracellular Transport ofTherapeutic Carriers to Human and Mouse ICAM-1 Via the Affinity Peptideγ3 and its Derivative Peptides 2γ3 and 3γ3

(A) The peptide γ3 was absorbed on the surface of 100 nm FITC-labeledpolystyrene carriers and the resulting products were incubated for 15min on nitrocellulose-immobilized albumin vs human or mouse ICAM-1chimeras containing ICAM-1-Ig domains 1 and 2 fused to human Fcsequence. The membranes were washed and analyzed by fluorescencemicroscopy to quantify the number of carriers bound per area. Data areexpressed relative to non-specific binding of γ3 carriers to albumincontrols (dotted line). A comparison to binding of anti-ICAM carriers isprovided (dashed line). (B) Binding of FITC-labeled polystyrene carrierscoated by absorption with the peptide γ3 and recombinant acidsphingomyelinase (ASM, a therapeutic enzyme) to activated HUVEC (bars)vs ICAM-1-negative 293 cells (dotted line) was quantified byfluorescence microscopy after 1 h incubation at 37° C., in the absenceor presence of excess γ3, anti-ICAM, γ3 scramble peptide, or mannose 6phosphate (M6P). Data is shown relative to HUVEC binding of γ3/ASMcarriers (134±11 particles/cell). (C) HUVEC lysosomes were labeled for 2h with Texas-red dextran at 37° C. Cells were then incubated for 30 minat 37° C. with FITC-labeled γ3/ASM carriers, non-bound carriers werewashed, and incubation was continued up to 1, 2, or 3 h. Co-localizationof FITC carriers with Texas-red dextran-lysosomes was quantified byfluorescence microscopy. (D) Comparison of lysosomal transport of 100 nmFITC-labeled polystyrene carriers coated with either ICAM-1-targetingpeptide γ3 vs its derivatives 2γ3 and 3γ3. (E) Activated HUVEC wereincubated for 30 min at 37° C. in the presence of FITC-labeled 2γ3polystyrene carriers after 15 min pre-treatment with 3 mM amiloride toblock ICAM-1-mediated endocytosis, or 0.1 μM PMA to activate PKC andpromote ICAM-1-mediated endocytosis. Co-localization of FITC 2γ3carriers with Texas red dextran-labeled lysosomes was assessed bymicroscopy 3 h post-internalization. Mean±SEM; n=2 assays. **p<0.005,***p<0.001, by student's t test.

Example 5 Efficient and Specific Binding and/or Intracellular Transportof Peptides or Peptide-Expressing Viruses to Human and Mouse ICAM-1 Viathe Affinity Peptides A1, B7, B8, B9, B10, and D6

(A) ELISA measurement of the binding of 7-mer peptide expressing phagelibrary to immobilized recombinant ICAM-1 (consisting of the two mostmembrane-distal Ig domains-1 and 2- of mouse ICAM-1 fused to human Fc orhFc), versus their binding to control hFc. (B) Relative specificity and(C) relative affinity of ICAM-1-targeting peptide phages, determined byELISA from the experiment in (A). (D) Relative binding of phagesexpressing 7-mer peptides to native ICAM-1 expressed on the surface ofHV5 mouse endothelial cells, either control or activated with TNFalphato mimic a disease phenotype. Binding was determined by microscopy usinga fluorescently-labeled antibody to the phage capsid. The line in thegraph indicates the level of binding of A1 phage to 293 cells, which arevoided of ICAM-1-expression. (E, F) Endocytosis (1 h, 37° C.) of phagesexpressing 7-mer peptides by control mouse endothelial cells (E) ormouse endothelial cells incubated in the presence of 3 mM amiloride (F)was estimated by microscopy after staining surface-located phages with aTexas red-labeled antibody to the viral capsid, following by cellpermeabilization and staining of all (surface and internalized) phagesusing a FITC-labeled antibody to the viral capsid. (G) Binding ofFITC-labeled anti-ICAM polystyrene nanocarriers to human endothelialcells was evaluated by fluorescence microscopy after incubation withcells in the absence (100% level) or presence of anti-ICAM antibody,ICAM-1-targeting synthetic peptides, or peptides with their scramblesequence (e.g., A1sc).

Example 6 In Vivo Pharmacokinetics and Biodistribution of TherapeuticCarriers Targeted to ICAM-1 Via the Affinity Peptide γ3 and itsDerivative Peptides 2γ3 and 3γ3

IgG/¹²⁵I-ASM polystyrene carriers vs γ3/¹²⁵I-ASM carriers, 2γ3/¹²⁵I-ASMcarriers or 3γ3/¹²⁵I-ASM carriers were injected iv in C57Bl/6 mice.Blood samples were taken at 15 min and 30 min after injection, and theorgans were collected 30 min after injection, to determine the presenceof ¹²⁵Iodine in the samples. (A) Percent of injected dose in circulationis shown for control IgG/¹²⁵I-ASM polystyrene carriers vs γ3/¹²⁵I-ASMcarriers. (B) Percent of injected dose in liver (an organ of unspecificclearance) and lung (and organ requiring targeting for accumulation) areshown. (C) Mice were injected with either free ¹²⁵I-ASM vs γ3/¹²⁵I-ASMcarriers, 2γ3/¹²⁵I-ASM carriers or 3γ3/¹²⁵I-ASM carriers, blood andorgans were collected 30 min after injection and the specificity indexof all samples was calculated. Specificity index=Localization Ratio ofthe carrier divided by the Localization Ratio of the free enzyme, wherethe Localization Ratio is the percent injected dose/gram in an organdivided by percent injected dose/gram in blood. (D) Liver, lung, andbrain uptake (30 min) of γ3/¹²⁵I-ASM polystyrene carriers compared toanti-ICAM/¹²⁵I-ASM carriers and control ¹²⁵I-ASM. (E) Lung targeting ofZγ3/¹²⁵I-ASM polystyrene carriers vs 2γ3-scramble/¹²⁵I-ASM carriers incontrol C57Bl/6 mice (bars), and Zγ3/¹²⁵I-ASM carriers in ICAM-1 knockout (ICAM-1 KO) mice (line across the bar on the left). Mean±SEM; n=3mice. (F) Transmission electron microscopy picture showing endocytosisof 2γ3/ASM PLGA carriers in lungs of mice (30 min post-injection).Asterisks=caveoli. EnC=Endothelial cell. EpC=Epithelial cell. Scalebar=100 nm.

Example 7 Therapeutic Effects of Cargoes Intracellularly Delivered toPatient Cells by γ3-Strategy Carriers

Skin fibroblasts from type A Niemann-Pick patients were incubatedovernight with BODIPY-FLC12-sphingomyelin to label sphingomyelinaccumulation in lysosomes in these cells. Cells were then incubated withcontrol media or treated for 5 h with recombinant ASM loaded on thesurface of 100 nm polystyrene carriers or poly-lactic co-glycolic acid(PLGA) carriers targeted to ICAM-1 via either anti-ICAM monoclonalantibody or the peptide γ3. The cells were then washed, fixed, andstained with filipin to label cholesterol, which also accumulatesintracellularly in Niemann-Pick disease. Samples were analyzed byfluorescence microscopy to determine the reduction of the levels ofsphingomyelin and cholesterol. The percent of accumulation of theselipids was compared to that of untreated diseased cells (100%accumulation). Mean±SEM, n>20 cells.

TABLE 1 Anti-ICAM/ASM γ3/ASM γ3/ASM (% no treatment) polystyrenepolystyrene PLGA Sphingomyelin 10 ± 1.7 10 ± 1.9 3 ± 1.3 Cholesterol  6± 1.0  5 ± 1.1 5 ± 0.7

Example 8 Design of a Chimeric Therapeutic Enzyme ContainingICAM-1-Targeting γ3, 2γ3 3γ3 or A1 Derivative Peptides

The coding sequence for the peptides γ3, 2γ3, 3γ3 or A1 can be formed byhybridization of the forward (F) and reverse (R) oligonucleotidesXmaI-ATG-γ3-SpeI, XmaI-ATG-2γ3-SpeI, XmaI-ATG-3γ3-SpeI,XmaI-ATG-B7-SpeI, XmaI-ATG-B8-SpeI, XmaI-ATG-B9-SpeI, XmaI-ATG-B10-SpeI,or XmaI-ATG-D6-SpeI, respectively. The coding sequence for spacerbetween these peptides and ASM can be formed by hybridization of theforward (F) and reverse (R) oligonucleotides SpeI-(Ser4Gly)2-EcoRI. ASMcoding sequence can be amplified by PCR from a plasmid containing ASMcDNA, using the forward (F) primer EcoRI-ASM and reverse (R) primerASM-XbaI. The resulting fragments can be cloned into pMT/BiP/V5-His (B)plasmid to obtain an ICAM-1-targeted ASM plasmid termed (B) (FIG. 7).(B) can be used for amplification in E. coli and expression in S2 cells,using single-site restriction enzyme digestion with XmaI, SpeI, EcoRI,and XbaI, respectively, and subsequent ligation.

Oligonucleotides: XmaI-ATG-γ3-SpeI-F (SEQ ID NO: 4)5′-CCGGGATGAATAATCAAAAGATTGTTAACCTGAAA GAGAAGGTAGCCCAGCTTGAAGCAA-3′XmaI-ATG-γ3-SpeI-R (SEQ ID NO: 5) 5′-CTAGTTGCTTCAAGCTGGGCTACCTTCTCTTTCAGGTTAACAATCTTTTGATTATTCATC-3′ XmaI-ATG-2γ3-SpeI-F (SEQ ID NO: 6)5′-CCGGGATGAATAATCAAAAGATTGTTAACATCAAAGA GAAGGTAGCCCAGATCGAAGCAA-3′XmaI-ATG-2γ3-SpeI-R (SEQ ID NO: 7)5′-CTAGTTTGCTTCGATCTGGGCTACCTTCTCTTTGA TGTTAACAATCTTTTGATTATTCATC-3′XmaI-ATG-3γ3-SpeI-F (SEQ ID NO: 8)5'-CCGGGATGAATAATCAAAAGCTTGTTAACATCAAAGA GAAGGTAGCCCAGATCGAAGCAA-3'XmaI-ATG-3γ3-SpeI-R (SEQ ID NO: 9)5′-CTAGTTGCTTCGATCTGGGCTACCTTCTCTTTGAT GTTAACAAGCTTTTGATTATTCATC-3′XmaI-ATG-A1-SpeI-F (SEQ ID NO: 20) 5′-CCGGGATGTACCCCGCCAGCTACCAGCGGA-3′XmaI-ATG-A1-SpeI-R (SEQ ID NO: 21) 5′-CTAGTCCGCTGGTAGCTGGCGGGGTACATCC-3′XmaI-ATG-D6-SpeI-F (SEQ ID NO: 22) 5′-CCGGGATGGGCTGCAAGCTGTGCGCCCAGA-3′XmaI-ATG-D6-SpeI-R (SEQ ID NO: 23) 5′-CTAGTCTGGGCGCACAGCTTGCAGCCCATC-3′XmaI-ATG-B7-SpeI-F (SEQ ID NO: 24) 5′-CCGGGATGTACCAGGCCACCCCCCTGCCCA-3′XmaI-ATG-B7-SpeI-R (SEQ ID NO: 25) 5′-CTAGTGGGCAGGGGGGTGGCCTGGTACATC-3′XmaI-ATG-B8-SpeI-F (SEQ ID NO: 26) 5′-CCGGGATGGGCAGCCTGCTGAGCGCCGCCA-3′XmaI-ATG-B8-SpeI-R (SEQ ID NO: 27) 5′-CTAGTGGCGGCGCTCAGCAGGCTGCCCATC-3′XmaI-ATG-B9-SpeI-F (SEQ ID NO: 28) 5′-CCGGGATGTTCAGCCCCCACAGCCGGACCA-3′XmaI-ATG-B9-SpeI-R (SEQ ID NO: 29) 5′-CTAGTGGTCCGGCTGTGGGGGCTGAACATC-3′XmaI-ATG-B10-SpeI-F (SEQ ID NO: 30) 5′-CCGGGATGTACCCCTTCCTGCCCACCGCCA-3′XmaI-ATG-B10-SpeI-R (SEQ ID NO: 31) 5′-CTAGTGGCGGTGGGCAGGAAGGGGTACATC-3′SpeI-(Ser4Gly)2-EcoRI-F (SEQ ID NO: 10)5′-ACTAGTTCTTCTTCTTCTGGCTCTTCTTCTTCTG GCGAATTC-3′SpeI-(Ser4Gly)2-EcoRI-R (SEQ ID NO: 11)5′-GAATTCGCCAGAAGAAGAAGAGCCAGAAGAAGAA GAACTAGT-3′ EcoRI-ASM-F(SEQ ID NO: 12) 5′-AATTCCCCCGCTACGGAGCGTCAC-3′ ASM-XbaI-R(SEQ ID NO: 13) 5′-CTAGACTAGCAAAACAGTGGCCTTG-3′

Example 9 In Vivo Brain Transport of Therapeutic Carriers Targeted toICAM-1 Via the Affinity Peptide 2γ3

Biocompatible PLGA carriers (100 nm diameter) were coated by surfaceabsorption with the ICAM-1-targeting peptide 2γ3 and the recombinantenzyme acid sphingomyelinase (ASM), taken as an example for atherapeutic cargo. The resulting 2γ3/ASM PLGA carriers were injectedintravenously in anesthetized C57Bl/6 mice. Three hours after injection,animals were euthanized under anesthesia, perfused and fixed though theleft ventricle of the heart under ventilation, and the brain wasisolated and processed for transmission electron microscopy.

FIG. 8 provides transmission electron microscopy pictures showingbiocompatible 2γ3/ASM PLGA carriers in brain of C57Bl/6 mice (3 hpost-injection). Scale bar=500 nm. FIG. 8(A) 2γ3/ASM PLGA carriers boundto the surface of (asterisk) or endocytosed within (arrows) vascularendothelial cells in the blood-brain barrier FIG. 8(B) 2γ3/ASM PLGAcarriers that have migrated across the blood-brain barrier into a regionof the brain close to the myelinated axon of a neuron.

Example 10 Inhibition of Inflammatory Leukocyte Transmigration AcrossEndothelial Cells by ICAM-1 Targeting Peptides

Human endothelial cells (human umbilical vein endothelial cells(HUVECs)) were grown until formation of a continuous confluent monolayerin a transwell filter located between upper and lower chambers. Thecells were then treated with tumor necrosis factor alpha to mimicinflammatory activation of endothelial cells and SDF1a was added to thelower chamber to generate a gradient to attract white blood cells, asduring inflammation. Lymphocytes isolated from human peripheral bloodwere activated with interleukin 2 and added to the upper chamber aboveendothelial cells in the absence (control) or presence ofICAM-1-targeting peptides A1, B7, B8, B9, B10, or D6. Albumin or apeptide of scrambled sequence were used as negative controls whichshould not block inflammatory transmigration of leukocytes acrossendothelial cells. The anti-ICAM antibody LB2, which is known to blockleukocyte adhesion to ICAM-1, was used as a positive control whichshould block such transmigration.

Leukocytes which transmigrated to the lower chamber below endothelialcells were counted to assess their transmigration. FIG. 9 provides agraph of the resulting leukocyte transmigration as a percentage of thecontrol. The peptides annotated with an asterisk in FIG. 9 indicate thatthe peptide was effective in statistically significant blockage ofleukocyte transmigration across endothelial cells. All ICAM-1-targetingpeptides tested were shown to inhibit transmigration of leukocytes in aspecific and effective manner.

Example 11 In Vivo Inhibition of Lodging of Fibrin Microemboli in theVasculature by ICAM-1 Targeting Peptides

C57Bl/6 mice were first injected intravenously with γ3 to block ICAM-1in the vascular endothelium in organs. Alternatively, injections ofsaline or anti-ICAM were used as controls. Fifteen minutes later themice were injected with fibrin clots (microemboli around 3-5 micrometer)generated in the lab by established procedures.

Following microemboli administration, the microemboli amounts weremeasured in a gamma counter and calculated as percent of injected dosein circulation or localization ratio (the percent injected dose/gram inan organ divided by percent injected dose/gram in blood). FIG. 10(A) isa graphical illustration of the amount of microemboli in circulation inblood, as evaluated at 1 min after injection and FIG. 10(B) is agraphical illustration of the amount of microemboli lodging in thevasculature of the kidneys and spleen at 15 min after injection.

In contrast to anti-ICAM, blocking of ICAM-1 with γ3 peptide causedfibrin microemboli to remain in circulation, attenuating microembolilodging in organs.

Example 12 ICAM-1 Expression in GI Epithelial Cells

Prior to examining specific binding of anti-ICAM carriers (model 100 nmdiameter fluorescent polystyrene-latex beads coated by surfaceabsorption with anti-ICAM) to GI epithelial cells, ICAM-1 expression wasverified. Human epithelial colorectal adenocarcinoma Caco-2 cell modelwas used. Both control cells as well as TNF-α-activated cells were usedto mimic normal versus pathologically altered conditions. ICAM-1 wasimmunolabeled with anti-ICAM followed by FITC goat anti-mouse IgG incontrol versus TNF-α-activated Caco-2 cells (fixed) and imaged byfluorescence microscopy. Immunofluorescence and microscopy imagingrevealed that Caco-2 cells expressed ICAM-1 under both conditions (FIG.11; magnification bar=10 μm.).

Example 13 Marked and Specific Binding of ICAM-1-Targeted Carriers in GIEpithelial Cells

Specific binding of anti-ICAM carriers to Caco-2 cells was tested andresults are provided in FIG. 12 (Magnification bar=10 μm. Data aremeans±S.E.M. (n≧20); *, p<0.001 by Student's t test). Binding of FITCcarriers (control IgG or anti-ICAM carriers) to fixed TNF-α-activatedCaco-2 cells was assessed by fluorescence microscopy. Anti-ICAMcarriers, not IgG carriers, were found to bind to TNF-α-activated Caco-2cells (FIG. 12A). The number of carriers bound per cell wasautomatically quantified by fluorescence microscopy and image analysis(FIG. 12B) revealed that binding of anti-ICAM carriers markedly exceededthat of non-specific IgG carriers by ˜62 fold (78±4 versus 1.3±0.1carriers/cell). These results verified the specificity of binding.

In addition, the Caco-2 cells were incubated with FITC anti-ICAMcarriers in the presence of non-specific IgG or anti-ICAM. It was seenthat presence of anti-ICAM antibodies free in solution greatly reducedbinding of anti-ICAM carriers to 2.4±0.3% of the control value with nocompetitor, whereas the presence of IgG did not affect binding (102±2.6%of the control value) (FIG. 12C). Effective competition by anti-ICAMfurther confirmed the specificity of binding of anti-ICAM carriers toCaco-2 cells.

Example 14 Kinetics of Binding and Endocytosis of ICAM-1-TargetedCarriers in GI Epithelial Cells

The efficiency of targeting of anti-ICAM carriers in Caco-2 cells wasdetermined. The amount of carriers bound per cell was quantified fromfluorescence images of fixed TNF-α-activated Caco-2 cells incubated withFITC-labeled anti-ICAM carriers. Fluorescence analysis (FIG. 13A)revealed that significant binding of anti-ICAM carriers to activatedCaco-2 cells occurred as early as 5 min (40.5±2.7 carriers bound percell). This amount nearly doubled by 30 min, which indicated a fastbinding rate and extent of carrier binding. Binding saturation occurredat 157 carriers bound per cell, demonstrating the high degree oftargeting and potential of this strategy.

Furthermore the ability of anti-ICAM carriers to be endocytosed byCaco-2 cells was examined. FITC-labeled anti-ICAM carriers were firstincubated with cells for 30 min to allow binding, then non-boundcarriers were washed and cells were incubated for varying times to allowcarrier uptake. After cell fixation, surface-bound carriers were stainedwith Texas Red secondary antibody. Fluorescence quantification expressedas % internalization revealed fast kinetics for carrier uptake(t_(1/2)=22 min and saturation at ˜1 h). Data are shown as means±S.E.M.(n≧20). Such analysis of uptake kinetics showed that FITC-labeledanti-ICAM carriers were internalized very rapidly by GI epithelial cells(50% internalization after 22 min) (FIG. 13B). Internalization washighly efficient: approximately 90% of the carriers bound to cells wereinternalized, which represents ˜60 carriers per cell.

Example 15 ICAM-1-Targeted Carriers are Internalized by Cam-MediatedEndocytosis in GI Epithelial Cells

The mechanism of uptake for Caco-2 cells treated with amiloride, a knowninhibitor of CAM-mediated endocytosis (Muro et al., 2003), was examined.TNF-α-treated Caco-2 cells were incubated with FITC-labeled anti-ICAMcarriers in the presence or absence of amiloride (1 h, 37° C.), andsurface-bound carriers were stained with TxR goat anti-mouse.Fluorescence microscopy revealed that amiloride reduced carrier uptaketo 20% of the control value (FIG. 14). Upper panels of FIG. 14A showtotal carriers associated to cells, whereas lower panels show thefraction of surface-bound, non-internalized carriers. (Magnificationbar=10 μm.) The fluorescence microscopy images were analyzed for theratio of internalized carriers to surface-bound carriers, expressed as %internalization and normalized to the control. (FIG. 14B; Data are shownas means±S.E.M. (n≧20). *, p<0.001 by Student's t test.)

The total amount of cell-associated carriers per cell was similar forboth amiloride-treated and control cells, which suggests that theeffects of this inhibitor in internalization are not due to effects onbinding. Therefore, CAM-mediated endocytosis is responsible for theuptake of anti-ICAM carriers in GI epithelial cells.

Example 16 Permeability Barrier in a Transwell GI Epithelial Model

In vitro permeability models designed to mimic physiological transportof materials from the apical (i.e. intestinal lumen) to the basolateral(i.e. systemic circulation) surface of cells were then used. Caco-2cells grown on porous permeable inserts are known to exhibit thephenotype of mature enterocytes, which include the presence of brushborder microvilli and tight junctions, dome formation, and production ofbrush border enzymes (Hidalgo, I. J., et al., Gastroenterology 96 (1989)736-749; Hidalgo, I. J. & Borchardt, R. T., Biochim. Biophys. Acta 1028(1990): 25-30.). Also, Caco-2 cells cultured on permeable membranes havebeen established in previous works as a model of the human intestine tostudy transepithelial drug transport (Hidalgo, I. J. & Borchardt, R. T.,Biochim. Biophys. Acta 1028 (1990): 25-30; Artusson, P., J. Pharma. Sci.79 (1990): 476-482.).

The Caco-2 model was confirmed as representative of the GI epithelium interms of the mentioned features. It was evaluated whether Caco-2 cellscould form a permeability barrier (indicative of the GI epithelialbarrier) under the particular growth conditions. In addition, thepresence of ICAM-1 was assessed to ensure nanocarrier targeting wouldoccur in this more physiologically relevant transport model.

In order to evaluate the proper growth conditions for a Caco-2 transportmodel, Caco-2 cells were cultured on permeable membrane inserts (0.4 μmpore) and the integrity of the permeability barrier was assessed usingtransepithelial electrical resistance (TEER) measured over time, wherebyincreased resistance to the flow of electrical current typicallysignifies closing of the epithelial junctions, a feature of healthy GIepithelium. It was verified whether TEER values indicative of apermeability barrier correlated with the formation of tight junctionsbetween Caco-2 cells. TEER values around or above 300 Ω×cm² indicateclosing of intercellular junctions and formation of the permabilitybarrier.

As shown in FIG. 15, cells presented low TEER values and did not formtight junctions within the first 5 days, but they showed a marked TEERincrease indicative of a permeability barrier (≧300 Ω×cm²) by day 14.Tight junctions formation was further confirmed by immunolabeling tightjunctions with anti-occludin followed by TxR donkey anti-goat IgG.Images were obtained using fluorescence microscopy. (Magnificationbar=10 μm.) In addition, treatment of cells with TNFα to mimicpathological activation of GI epithelial cells did not disrupt thispermeability barrier. Therefore, this represents a good model to testtransepithelial transport of ICAM-1-targeted carriers across the GI.

Example 17 ICAM-1 Expression in Differentiated GI Epithelial Cells

In order to ensure that targeting of anti-ICAM carriers will also occurin such polarized Caco-2 cells, expression of ICAM-1 was examined.Resting and TNF-α-activated Caco-2 cells were grown on permeablemembrane inserts. Once confluence was confirmed by TEER, the cells wereimmunolabeled with either anti-ICAM or control anti-PECAM, followed byFITC-labeled goat anti-mouse IgG. As expected, immunofluorescence withanti-ICAM FIG. 16A) confirmed the expression of ICAM-1 in Caco-2 cellsgrown to confluence and forming a permeability barrier on transwellfilters. A single cell and its microvilli are marked in FIG. 16(Magnification bar=10 μm), representative of differentiated Caco-2cells. As a control, the absence of platelet endothelial cell adhesionmolecule-1 (PECAM-1), a related cell adhesion molecule which is onlyexpressed by endothelial cells, confirmed the specificity of ICAM-1expression (FIG. 16B). These experiments further revealed expression ofICAM-1 in microvilli surfaces on Caco-2 cells. Surprisingly, in thismore physiological model of GI epithelial cells, ICAM-1 expression wasvery high in control cells (comparable to pathologically activatedcells), supporting the potential of this strategy for drug delivery inboth settings.

Example 18 Transport of ICAM-1-Targeting Carriers Across GI EpithelialMonolayers

Transport of anti-ICAM carriers across Caco-2 cell layers was examinedin cells growing on transwell filters by addition of carriers to theapical/top chamber and collection of transported carriers from thebasolateral/bottom chamber. Caco-2 monolayers grown on permeablemembrane inserts were incubated with ¹²⁵I-anti-ICAM or control ¹²⁵I-IgGcarriers added to the apical chamber.

Radioactivity in the basolateral fraction was measured at each timepoint and converted to the amount of anti-ICAM carriers transported percell. As shown in FIG. 17A, transport of anti-ICAM carriers from theapical to the basolateral side of Caco-2 cells was highly relevant inabsolute values, which markedly increased from ˜170 anti-ICAM carriersbeing transported per cell at 30 min up to ˜6,000 carriers per cell at48 h. This level of transport is equivalent to transport of as much as3×10⁷ carriers per mm² epithelial tissue. These results demonstrate thesurprising and unexpected high extent to which anti-ICAM carriers aretransported across Caco-2 cells.

In addition, transport was specific as shown by the fact that only afraction of control IgG carriers were transported across the Caco-2monolayer even after very long incubation times FIG. 17B shows transportof IgG carriers normalized to the transport of anti-ICAM carriers at 24h. (Data are shown as means±S.E.M. (n=4 wells). *, p<0.001 by Student'st test.)

Example 19 Rate of Carrier Transport Across GI Epithelial Cells

Caco-2 cells grown on permeable membrane inserts were incubated with¹²⁵I-anti-ICAM carriers over time. Percent of transported carriers wascalculated as the ratio of carriers in the basolateral fraction to thatin the combined basolateral and cell fractions. FIG. 18A shows thepercent of transported carriers with respect to the total amount ofcarriers associated to the cells. This parameter demonstrates a veryrapid rate of transport, e.g., 34% after only 30 min, 43% after 1 h, anda maximum value of 75% of carrier transport at 48 h, indicating that themajority of anti-ICAM carriers that bind to cells at the apical side aretransported across the cell body and secreted at the abluminal side.

The apparent permeability coefficient (P_(app),) is another parameterthat indicates rate of transport and allows comparison of permeabilitybetween different solutes. Apparent permeability coefficients werederived from rates of transport of ¹²⁵I-bovine serum albumin (BSA)versus ¹²⁵I-anti-ICAM carriers (24 h). The permeability coefficient ofanti-CAM carriers was compared to that of albumin, a serum protein knownto maintain the oncotic pressure by being capable of transport acrosscellular layers into the parenchyma of tissues (Rezai, K. A., et al.,Graefes. Arch. Clin. Exp. Opthalmol. 235 (1997): 48-55; Savin, V. J. etal., J Am Soc Nephrol 3 (1992): 1260-1269.). As shown in FIG. 18B, therelative permeability of ¹²⁵I-anti-ICAM carriers was 4.6 fold greaterthan that of ¹²⁵I-albumin (1.4±0.1×10⁷ cm-s⁻¹ versus 0.3±0.02×10⁷cm-s⁻¹), suggesting that anti-ICAM carriers are involved in fasttransport with respect to a standard known for passive transport. (Dataare shown as means±S.E.M. (n=4 wells). *, p<0.001 by Student's t test.)

Example 20 Transport of ICAM-1-Targeting Carriers Across GI EpithelialCells by Cam-Mediated Trans Cytosis

The mechanism of transport of anti-ICAM carriers across Caco-2 cells wasexamined. Paracellular transport operates via the opening of celljunctions, which can be indicated by a drop in transepithelialelectrical resistance. TEER was measured during transport of¹²⁵I-anti-ICAM carriers to assess paracellular transport, indicated bydecreased TEER. Control TEER measured prior to transport is marked asthe interval of S.E.M (two lines). Results are provided in FIG. 19A,where it can be seen that the transport of anti-ICAM carriers acrossCaco-2 cells over time did not significantly lowered TEER with respectto pre-transport measurements (2γ3±33 vs 290±24 Ω-cm², respectively).Maintenance of high epithelial resistance suggested that anti-ICAMcarriers do not influence opening of intercellular junctions over thecourse of transport and, hence, it is likely that they do not compromisethe integrity of Caco-2 cell monolayers.

In addition, because albumin utilizes paracellular as well ascaveolin-mediated transcellular transport, it was used as a marker toindicate whether anti-ICAM carriers are transported via either of thesemechanisms. The amount of transported ¹²⁵I-albumin (bovine serum albuminor BSA) during simultaneous transport of anti-ICAM carriers (3 h) wasnormalized to the control (absence of anti-ICAM carriers). Radioisotopetracing confirmed that the transport of anti-ICAM carriers did notincrease the quantity of ¹²⁵I-albumin transported (FIG. 19B), implyingthe carriers do not open tight junctions to allow greater permeabilityof albumin or are transported via caveolin-mediated pathway.

Amiloride-treated versus control Caco-2 cells were incubated with¹²⁵I-anti-ICAM carriers for 3 h. Radioisotope analysis was conducted todetermine the amount of transported carriers. The results furtherdemonstrated that amiloride, a drug that inhibits CAM-mediatedendocytosis, reduced the transport of ¹²⁵I-anti-ICAM carriers by about50% (FIG. 19B). Therefore, these results support a role for CAM-mediatedendocytosis in the transcytosis of anti-ICAM carriers by Caco-2 cells.(Data are shown as means±S.E.M. (n=4 wells). *, p<0.01 by Student's ttest.)

Transepithelial transport involving CAM endocytosis has not yet beenidentified or characterized. Although opening of tight junctions maylead to faster transport, at the same time paracellular pathways poses athreat to epithelial monolayer integrity and its permeability barrier.Hence, these results seem to indicate the potential safety of transportof anti-ICAM carriers in the gastrointestinal epithelium.

Example 21 Cam-Mediated Transport of ICAM-1-Targeting Moieties in GIEpithelial Cells

Antibodies to ICAM-1 have been shown to induce therapeutic benefits uponbinding to their target (Takei, Y. et al., Transplant Proc 28 (1996):1103-1105; Kavanaugh, A. F., et al., Arthritis Rheum 40 (1997): 849-853;Hallahan, D. E. & Virudachalam, S. PNAS. 94 (1997): 6432-6437). Inaddition, targeting moieties alone, including anti-ICAM, can serve asaffinity carriers for drugs.

In order to assess the capability of ant-ICAM as therapeutics oraffinity carriers, the binding and potential endocytic uptake ofanti-ICAM in Caco-2 cells was tested. Upon 30 min and 1 h incubation(37° C.) in TNF-α-stimulated and control Caco-2 cells, surface-boundanti-ICAM was immunolabeled with TxR goat anti-mouse IgG, following cellpermeabilization and immunolabeling of both bound and internalizedanti-ICAM fractions with FITC goat anti-mouse IgG. Percentinternalization was automatically quantified from the images usingfluorescence microscopy (FIG. 20A). Similar expriments were conducted inthe absence (control) or presence of amiloride, a drug that inhibitsCAM-mediated endocytosis. Percent internalized anti-ICAM wasautomatically analyzed by fluorescence microscopy (FIG. 20B).

In previous studies describing CAM-mediated endocytosis, monomeric (asingle copy of) anti-ICAM allowed binding but not internalization in thecase of endothelial cells. Surprisingly, fluorescence microscopy ofsurface versus internalized fractions showed that anti-ICAM (non-coupledto carriers) induced endocytosis in both control and pathological modelsof Caco-2 cells. Moreover, the levels of endocytosed anti-ICAM at werecomparable in control and TNF-α-activated Caco-2 cells, indicatingsimilar uptake kinetics between the two conditions. The mechanism ofanti-ICAM uptake was inferred by treatment with amiloride, whichindicated that uptake of anti-ICAM by Caco-2 cells also operates viaCAM-mediated endoytosis even in the case of non-multivalent binding toICAM-1. (FIG. 20; Data are shown as means±S.E.M. (n≧50). *, p<0.001 byStudent's t test.)

Example 22 Peptides as Targeting Moieties for GI Delivery

Similar experiments using short peptides derived from fibrin and/orpeptides identified by phage display as targeting moieties for specifictargeting of ICAM-1 on GI epithelial cells are expected to demonstratethat compounds containing such peptides as targeting moieties will besimilarly transported across the GI epithelial layer.

Such anticipated results are based upon the prior examples demonstratinguptake of the peptide-containing compounds by vascular endothelial cellsand transport across the blood-brain barrier (Example 9; FIG. 8).

Similar uptake by GI epithelial cells and observation of transportacross the GI epithelial layer are expected.

While the invention has been has been described herein in reference tospecific aspects, features and illustrative embodiments of theinvention, it will be appreciated that the utility of the invention isnot thus limited, but rather extends to and encompasses numerous othervariations, modifications and alternative embodiments, as will suggestthemselves to those of ordinary skill in the field of the presentinvention, based on the disclosure herein. Correspondingly, theinvention as hereinafter claimed is intended to be broadly construed andinterpreted, as including all such variations, modifications andalternative embodiments, within its spirit and scope.

What is claimed is:
 1. A composition for oral administration to asubject, the composition comprising: a) a targeting moiety comprising ananti-ICAM antibody; and b) an agent, wherein the targeting moietyrecognizes and binds to ICAM-1 on a gastrointestinal epithelial cell andthe composition is transported across the gastrointestinal epithelium.2. The composition of claim 1, wherein the agent comprises any of aresearch probe, an analytical probe, a reporter probe, a molecularprobe, a diagnostic agent, a therapeutic agent, a biologically activeagent, a research agent, an analytical agent, an imaging agent, amonitoring agent, an enzyme, a protein, a peptide, a nucleic acid, alipid, a sugar, a hormone, a lipoprotein, a chemical, a virus, abacterium, a cell, a biosensor, a marker, an antibody and a ligand. 3.The composition of claim 2, wherein the agent comprises a lysosomalenzyme.
 4. The composition of claim 2, wherein the agent comprises anyof the enzymes involved in Pompe Disease, GM1 gangliosidosis, Tay-Sachsdisease, GM2 gangliosidosis, Sandhoff disease, Fabry disease, Gaucherdisease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pickdisease type A, Niemann-Pick disease type B, Niemann-Pick disease typeC, Niemann-Pick disease type D, Farber disease, Wolman disease, HurlerSyndrome, Scheie Syndrome, Hurler-Scheie Syndrome, Hunter Syndrome,Sanfilippo A Syndrome, Sanfilippo B Syndrome, Sanfilippo C Syndrome,Sanfilippo D Syndrome, Morquio A disease, Morquio B disease,Maroteaux-Lamy disease, Sly Syndrome, α-mannosidosis, β-mannosidosis,fucosidosis, aspartylglucosaminuria, sialidosis, mucolipidosis II,mucolipidosis III, mucolipidosis IV, Goldberg Syndrome, Schindlerdisease, cystinosis, Salla disease, infantile sialic acid storagedisease, Batten disease, infantile neuronal ceroid lipofuscinosis, andprosaposin.
 5. The composition of claim 1, further comprising a deliverycarrier for transport of the targeting moiety and agent to thegastrointestinal epithelial cell.
 6. The composition of claim 5, whereinthe delivery carrier comprises a natural virus or derived viral-likeparticle, dendrimer, carbon nanoassembly, liposome, a polymer carrier, amicrobubble, a paramagnetic particle, a ferromagnetic particle, aself-assembled polymer, a polymersome, a filomicelle, a micelle, a microparticle or nanoparticle, an albumin particle, and/or a lipoprotein. 7.The composition of claim 1, further comprising a protective agenteffective to protect the targeting moiety and agent from degradationprior to binding of ICAM-1.
 8. The composition of claim 7, wherein theprotective agent comprises a polymer.
 9. The composition of claim 8,wherein the polymer is in a gel form.
 10. The composition of claim 8wherein the polymer comprises chitosan.
 11. The composition of claim 1,further comprising a second targeting moiety, effective to target acell, tissue or organ after transport across the gastrointestinalepithelium, wherein the second targeting moiety recognizes and binds toa target on the cell, tissue or organ, and is effective to deliver theagent to the cell, tissue or organ.
 12. The composition of claim 11,wherein the second targeting moiety comprises a moiety selected from SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, antibody, an aptamer, anucleic acid, a peptide, a carbohydrate, a lipid, a vitamin, a toxin, acomponent of a microorganism, a hormone, and a receptor ligand.
 13. Thecomposition of claim 12, wherein the second targeting moiety comprisesSEQ ID NO:1.
 14. The composition of claim 12, wherein the secondtargeting moiety comprises a moiety selected from an aptamer, a nucleicacid, a peptide, a carbohydrate, a lipid, a vitamin, a toxin, acomponent of a microorganism, a hormone, and a receptor ligand.